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- PDB-8c0b: CryoEM structure of Aspergillus nidulans UTP-glucose-1-phosphate ... -

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Basic information

Entry
Database: PDB / ID: 8c0b
TitleCryoEM structure of Aspergillus nidulans UTP-glucose-1-phosphate uridylyltransferase
ComponentsUTP--glucose-1-phosphate uridylyltransferase
KeywordsSUGAR BINDING PROTEIN / NDP-sugar pyrophosphorylases / UDP-Glc pyrophosphorylases / cell wall biosynthesis / Aspergillus nidulans.
Function / homology
Function and homology information


(1->6)-beta-D-glucan biosynthetic process / sporulation / UTP-glucose-1-phosphate uridylyltransferase / UTP:glucose-1-phosphate uridylyltransferase activity / trehalose biosynthetic process / UDP-alpha-D-glucose metabolic process / glycogen biosynthetic process / glycogen metabolic process / cytoplasm
Similarity search - Function
UTP--glucose-1-phosphate uridylyltransferase / UDPGP family / UTP--glucose-1-phosphate uridylyltransferase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
UTP--glucose-1-phosphate uridylyltransferase
Similarity search - Component
Biological speciesAspergillus nidulans FGSC A4 (mold)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.98 Å
AuthorsHan, X. / D Angelo, C. / Otamendi, A. / Cifuente, J.O. / de Astigarraga, E. / Ochoa-Lizarralde, B. / Grininger, M. / Routier, F.H. / Guerin, M.E. / Fuehring, J. ...Han, X. / D Angelo, C. / Otamendi, A. / Cifuente, J.O. / de Astigarraga, E. / Ochoa-Lizarralde, B. / Grininger, M. / Routier, F.H. / Guerin, M.E. / Fuehring, J. / Etxebeste, O. / Connell, S.R.
Funding support Spain, 1items
OrganizationGrant numberCountry
Ministry of Economy and Competitiveness (MINECO)PID2021-122705OB-I00 Spain
CitationJournal: mBio / Year: 2023
Title: CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from reveals key conformations for activity regulation and function.
Authors: Xu Han / Cecilia D'Angelo / Ainara Otamendi / Javier O Cifuente / Elisa de Astigarraga / Borja Ochoa-Lizarralde / Martin Grininger / Francoise H Routier / Marcelo E Guerin / Jana Fuehring / ...Authors: Xu Han / Cecilia D'Angelo / Ainara Otamendi / Javier O Cifuente / Elisa de Astigarraga / Borja Ochoa-Lizarralde / Martin Grininger / Francoise H Routier / Marcelo E Guerin / Jana Fuehring / Oier Etxebeste / Sean R Connell /
Abstract: Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic ...Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus , including , the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for (UGP). To understand the molecular basis of UGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native UGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. UGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for UGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.
History
DepositionDec 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_admin
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 / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Jul 24, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: UTP--glucose-1-phosphate uridylyltransferase
B: UTP--glucose-1-phosphate uridylyltransferase
C: UTP--glucose-1-phosphate uridylyltransferase
D: UTP--glucose-1-phosphate uridylyltransferase
E: UTP--glucose-1-phosphate uridylyltransferase
F: UTP--glucose-1-phosphate uridylyltransferase
G: UTP--glucose-1-phosphate uridylyltransferase
H: UTP--glucose-1-phosphate uridylyltransferase


Theoretical massNumber of molelcules
Total (without water)461,0778
Polymers461,0778
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
UTP--glucose-1-phosphate uridylyltransferase


Mass: 57634.672 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Aspergillus nidulans FGSC A4 (mold)
References: UniProt: C8VK50, UTP-glucose-1-phosphate uridylyltransferase

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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: UTP--glucose-1-phosphate uridylyltransferase / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Aspergillus nidulans FGSC A4 (mold)
Buffer solutionpH: 7.8
Details: 50 mM Tris, 200 mM NaCl, 2 mM Beta-mercaptoethanol, pH 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
2200 mMsodium chlorideNaCl1
32 mMbeta-mercaptoethanol1
SpecimenConc.: 0.18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_4788refinement
PHENIXdev_4788refinement
UCSF ChimeraX1.5/v9model building
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126375 / Symmetry type: POINT

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