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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)
KeywordsTRANSFERASE / Glycosyltransferase / Complex
Function / homology
Function and homology information


alpha,alpha-trehalose-phosphate synthase complex (UDP-forming) / trehalose-phosphatase activity / trehalose biosynthetic process / hexosyltransferase activity / trehalose metabolism in response to stress / cellular response to heat / cytosol
Similarity search - Function
Glycosyl transferase, family 20 / Glycosyltransferase family 20
Similarity search - Domain/homology
6-O-phosphono-alpha-D-glucopyranose / URIDINE-5'-DIPHOSPHATE / Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
Similarity search - Component
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: 2024
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 / Jennifer L Tenor / Dena L Toffaletti / Ziqiang Guan / John R Perfect / 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 annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of ...Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies residues required for substrate-binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), 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 density maps. We determined that the IDD is not required for Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. , 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.
SIGNIFICANCE STATEMENT: Fungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to ...SIGNIFICANCE STATEMENT: Fungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to transition from the environment to the human host. Enzymes in the trehalose biosynthesis pathway are absent in humans and, therefore, are potentially significant targets for novel antifungal therapeutics. One enzyme in the trehalose biosynthesis is trehalose-6-phosphate synthase (Tps1). Here, we describe the cryo-electron microscopy structures of the CnTps1 homo-tetramer in the unliganded form and in complex with a substrate and a product. These structures and subsequent biochemical analysis reveal key details of substrate-binding residues and substrate specificity. These structures should facilitate structure-guided design of inhibitors against CnTps1.
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
Revision 1.2Jun 26, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _citation.year / _em_admin.last_update

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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
Water00
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)


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


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 FIELD / 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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