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- PDB-8fhw: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphat... -

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Basic information

Entry
Database: PDB / ID: 8fhw
TitleCryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
ComponentsAlpha,alpha-trehalose-phosphate synthase (UDP-forming)Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
KeywordsTRANSFERASE / Glycosyltransferase / Complex
Function / homologyGlycosyl transferase, family 20 / Glycosyltransferase family 20 / trehalose biosynthetic process / hexosyltransferase activity / 6-O-phosphono-alpha-D-glucopyranose / URIDINE-5'-DIPHOSPHATE / Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
Function and homology information
Biological speciesCryptococcus neoformans var. grubii H99 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWashington, E.J. / Brennan, R.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1P01AI104533-01A1 United States
CitationJournal: bioRxiv / Year: 2023
Title: Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen : a target for novel antifungals.
Authors: Erica J Washington / Ye Zhou / Allen L Hsu / Matthew Petrovich / Mario J Borgnia / Alberto Bartesaghi / Richard G Brennan /
Abstract: Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million deaths worldwide each year. Yet the arsenal of antifungal therapeutics remains limited and is in dire ...Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million deaths worldwide each year. Yet the arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional fungal-specific biosynthetic pathways. One such pathway involves the biosynthesis of trehalose. Trehalose is a non-reducing disaccharide composed of two molecules of glucose that is required for pathogenic fungi, including and , to survive in their human hosts. Trehalose biosynthesis is a two-step process in fungal pathogens. Trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate (T6P). Subsequently, trehalose-6-phosphate phosphatase (Tps2) converts T6P to trehalose. The trehalose biosynthesis pathway has been identified as a top candidate for novel antifungal development based on quality, occurrence, specificity, and assay development. However, there are currently no known antifungal agents that target this pathway. As initial steps to develop Tps1 from (CnTps1) as a drug target, we report the structures of full-length CnTps1 and CnTps1 in complex with uridine diphosphate (UDP) and glucose-6-phosphate (G6P). Both CnTps1 structures are tetramers and display D2 (222) molecular symmetry. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies key residues required for substrate-binding, which are conserved amongst other Tps1 enzymes, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), encompassing residues M209 to I300, which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in the density maps. Although, activity assays revealed that the highly conserved IDD is not required for catalysis , we hypothesize that the IDD is required for Tps1-dependent thermotolerance and osmotic stress survival. Characterization of the substrate specificity of CnTps1 revealed that UDP-galactose, an epimer of UDP-glucose, is a very poor substrate and inhibitor of the enzyme and highlights the exquisite substrate specificity of Tps1. , these studies expand our knowledge of trehalose biosynthesis in and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.
History
DepositionDec 15, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
B: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
C: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
D: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)309,91912
Polymers307,2624
Non-polymers2,6578
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Alpha,alpha-trehalose-phosphate synthase (UDP-forming) / Alpha,alpha-trehalose-phosphate synthase (UDP-forming)


Mass: 76815.500 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: UDP G6P
Source: (gene. exp.) Cryptococcus neoformans var. grubii H99 (fungus)
Gene: CNAG_05292 / Plasmid: pMCSG7 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: J9VJZ1
#2: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE / Uridine diphosphate


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#3: Sugar
ChemComp-G6P / 6-O-phosphono-alpha-D-glucopyranose / ALPHA-D-GLUCOSE-6-PHOSPHATE / 6-O-phosphono-alpha-D-glucose / 6-O-phosphono-D-glucose / 6-O-phosphono-glucose


Type: D-saccharide, alpha linking / Mass: 260.136 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H13O9P / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
a-D-Glcp6PO3IUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
Has ligand of interestY

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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: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.307 MDa / Experimental value: YES
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41(DE3) / Plasmid: pMCSG7
Buffer solutionpH: 7.5
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 854608 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 48.64 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004814008
ELECTRON MICROSCOPYf_angle_d0.538819072
ELECTRON MICROSCOPYf_chiral_restr0.0432140
ELECTRON MICROSCOPYf_plane_restr0.00332400
ELECTRON MICROSCOPYf_dihedral_angle_d15.29814876

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