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- PDB-5h18: Crystal structure of catalytic domain of UGGT (UDP-glucose-bound ... -

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Basic information

Entry
Database: PDB / ID: 5h18
TitleCrystal structure of catalytic domain of UGGT (UDP-glucose-bound form) from Thermomyces dupontii
ComponentsUGGT
KeywordsTRANSFERASE / ENDOPLASMIC RETICULUM / QUALITY CONTROL / GLUCOSYLTRANSFERASE / FOLDING SENSOR
Function / homology
Function and homology information


UDP-glucose:glycoprotein glucosyltransferase activity / sporulation / protein N-linked glycosylation via asparagine / unfolded protein binding / nucleotide binding / endoplasmic reticulum / metal ion binding
Similarity search - Function
Glucosyltransferase 24, catalytic domain / Glucosyltransferase 24 / UDP-glucose:Glycoprotein Glucosyltransferase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
URIDINE-5'-DIPHOSPHATE-GLUCOSE / UGGT
Similarity search - Component
Biological speciesThermomyces dupontii (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.4 Å
AuthorsSatoh, T. / Zhu, T. / Toshimori, T. / Kamikubo, H. / Uchihashi, T. / Kato, K.
Funding support Japan, 3items
OrganizationGrant numberCountry
MEXT25102001, 25102008 Japan
PRESTO, JST13417569 Japan
CitationJournal: Sci Rep / Year: 2017
Title: Visualisation of a flexible modular structure of the ER folding-sensor enzyme UGGT.
Authors: Tadashi Satoh / Chihong Song / Tong Zhu / Takayasu Toshimori / Kazuyoshi Murata / Yugo Hayashi / Hironari Kamikubo / Takayuki Uchihashi / Koichi Kato /
Abstract: In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates ...In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates displayed on the protein surface serve as quality tags. The ER folding-sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a gatekeeper in the ER quality control system by specifically catalysing monoglucosylation onto incompletely folded glycoproteins, thereby enabling them to interact with lectin-chaperone complexes. Here we characterise the dynamic structure of this enzyme. Our crystallographic data demonstrate that the sensor region is composed of four thioredoxin-like domains followed by a β-rich domain, which are arranged into a C-shaped structure with a large central cavity, while the C-terminal catalytic domain undergoes a ligand-dependent conformational alteration. Furthermore, small-angle X-ray scattering, cryo-electron microscopy and high-speed atomic force microscopy have demonstrated that UGGT has a flexible modular structure in which the smaller catalytic domain is tethered to the larger folding-sensor region with variable spatial arrangements. These findings provide structural insights into the working mechanism whereby UGGT operates as a folding-sensor against a variety of glycoprotein substrates through its flexible modular structure possessing extended hydrophobic surfaces for the recognition of unfolded substrates.
History
DepositionOct 8, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 27, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2017Group: 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 23, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: UGGT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,7964
Polymers35,0981
Non-polymers6983
Water4,738263
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)45.470, 47.240, 129.780
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein UGGT


Mass: 35097.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces dupontii (fungus) / Plasmid: pCold-I / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2D0TCJ6*PLUS
#2: Chemical ChemComp-UPG / URIDINE-5'-DIPHOSPHATE-GLUCOSE / URIDINE-5'-MONOPHOSPHATE GLUCOPYRANOSYL-MONOPHOSPHATE ESTER


Mass: 566.302 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H24N2O17P2
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 263 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.03 Å3/Da / Density % sol: 39.6 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 9 / Details: 24% PEG 3350, 100 mM Tris-HCl (pH 9.0)

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NW12A / Wavelength: 0.97934 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: May 23, 2016
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97934 Å / Relative weight: 1
ReflectionResolution: 1.4→47.27 Å / Num. obs: 56063 / % possible obs: 100 % / Observed criterion σ(I): -3 / Redundancy: 7.1 % / CC1/2: 0.994 / Rmerge(I) obs: 0.14 / Net I/σ(I): 7.7
Reflection shellResolution: 1.4→1.48 Å / Redundancy: 7 % / Rmerge(I) obs: 0.6 / Mean I/σ(I) obs: 2.6 / CC1/2: 0.834 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0155refinement
iMOSFLMdata reduction
SCALAdata scaling
SHELXCDphasing
RefinementMethod to determine structure: SAD / Resolution: 1.4→20 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.964 / SU B: 2.074 / SU ML: 0.037 / Cross valid method: THROUGHOUT / ESU R: 0.06 / ESU R Free: 0.055 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.174 2756 4.9 %RANDOM
Rwork0.142 ---
obs0.144 53176 99.9 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 17.973 Å2
Baniso -1Baniso -2Baniso -3
1-0.01 Å20 Å20 Å2
2--0.39 Å20 Å2
3----0.4 Å2
Refinement stepCycle: 1 / Resolution: 1.4→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2271 0 43 263 2577
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0192422
X-RAY DIFFRACTIONr_bond_other_d0.0020.022273
X-RAY DIFFRACTIONr_angle_refined_deg1.5921.9813293
X-RAY DIFFRACTIONr_angle_other_deg0.95835232
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9195284
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.53523120
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.16215384
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.9991520
X-RAY DIFFRACTIONr_chiral_restr0.0920.2345
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0212668
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02596
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.5161.541100
X-RAY DIFFRACTIONr_mcbond_other1.5131.5371099
X-RAY DIFFRACTIONr_mcangle_it1.8682.3091375
X-RAY DIFFRACTIONr_mcangle_other1.8682.3111376
X-RAY DIFFRACTIONr_scbond_it1.8431.8141322
X-RAY DIFFRACTIONr_scbond_other1.841.8141322
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.2172.6241912
X-RAY DIFFRACTIONr_long_range_B_refined2.96719.4182861
X-RAY DIFFRACTIONr_long_range_B_other2.83318.9552803
X-RAY DIFFRACTIONr_rigid_bond_restr1.98434695
X-RAY DIFFRACTIONr_sphericity_free22.5855146
X-RAY DIFFRACTIONr_sphericity_bonded7.65954738
LS refinement shellResolution: 1.4→1.436 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.264 187 -
Rwork0.203 3863 -
obs--99.98 %

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