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- EMDB-29172: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphat... -

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Basic information

Entry
Database: EMDB / ID: EMD-29172
TitleCryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
Map dataCryptococcus neoformans trehalose-6-phosphate synthase (Tps1) homotetramer bound to UDP and G6P
Sample
  • Complex: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
    • Protein or peptide: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: 6-O-phosphono-alpha-D-glucopyranose
KeywordsGlycosyltransferase / Complex / TRANSFERASE
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
Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
Similarity search - Component
Biological speciesCryptococcus neoformans var. grubii H99 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWashington EJ / Brennan RG
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29172.png

Downloads & links

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Map

FileDownload / File: emd_29172.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryptococcus neoformans trehalose-6-phosphate synthase (Tps1) homotetramer bound to UDP and G6P
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 1.25
Minimum - Maximum-2.94728 - 6.0290866
Average (Standard dev.)-0.0022126662 (±0.18146421)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryptococcus neoformans trehalose-6-phosphate synthase (Tps1) homotetramer bound to...

Fileemd_29172_half_map_1.map
AnnotationCryptococcus neoformans trehalose-6-phosphate synthase (Tps1) homotetramer bound to UDP and G6P Half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryptococcus neoformans trehalose-6-phosphate synthase (Tps1) homotetramer bound to...

Fileemd_29172_half_map_2.map
AnnotationCryptococcus neoformans trehalose-6-phosphate synthase (Tps1) homotetramer bound to UDP and G6P Half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphat...

EntireName: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
Components
  • Complex: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
    • Protein or peptide: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: 6-O-phosphono-alpha-D-glucopyranose

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Supramolecule #1: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphat...

SupramoleculeName: Cryo-EM structure of Cryptococcus neoformans trehalose-6-phosphate synthase homotetramer in complex with uridine diphosphate and glucose-6-phosphate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Molecular weightTheoretical: 307 KDa

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

MacromoleculeName: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)
type: protein_or_peptide / ID: 1 / Details: UDP G6P / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Molecular weightTheoretical: 76.8155 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNAMTTMSN DIPNSPTSTS FTGTFSPAAT AANTAANART SDAPSPTTSS SGPKLETSKE QRLIVVSNR LPVTISKDDN GEYHFKMSSG GLVSALSGCK KTMSFTWIGW PGKDIPMQDR ETVNRRLLDE YNCYPVYLSD E LADSHYNG ...String:
MHHHHHHSSG VDLGTENLYF QSNAMTTMSN DIPNSPTSTS FTGTFSPAAT AANTAANART SDAPSPTTSS SGPKLETSKE QRLIVVSNR LPVTISKDDN GEYHFKMSSG GLVSALSGCK KTMSFTWIGW PGKDIPMQDR ETVNRRLLDE YNCYPVYLSD E LADSHYNG FSNSILWPLF HYHPGEMNFD AAHWLAYREA NMRFADVVSS LVQAGDMVWV QDYHLMLLPM LLRSMITGES AQ GEMVRQE LGRVKEGVDD TVVKEVLKMG PGVAQAEDEG VEMLDDVEEE GGEMDVKSSP KRPHYARGMS TFQKQELVAK EKG KEGIRI GFFLHTPFPS SEIYRILPVR REILLGVLQC DLIGFHTYDY ARHFLSSCTR ILGLETQPNG IEFDGRYCQV GTFP IGIDP NQFIEGLQKE SIVKRLRSLE ARFEGVKVII GVDRLDYIKG IPQKLQALET FLTQHPEWIG KVVLVQLAIP SRQDV EEYQ DLRACVNELV GRINGRFGTV ESVPIHYMHK SVPFEELTAM YALADACLVT STRDGMNLVA YEYISSQAER HGSMIL SEF AGAAQSFNGS LLINPWDVQS TADAINQALT LSPQQRKTNW QKLFNYVSKY TAEAWGVSFV NELNRLSGQR PSGPTGL AG RRKSGSLSRT SSKASIQRRK SSQSGIVTGL GAAAGAAVNW AQAQVQGGSQ T

UniProtKB: Alpha,alpha-trehalose-phosphate synthase (UDP-forming)

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Macromolecule #2: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

ChemComp-UDP:
URIDINE-5'-DIPHOSPHATE / UDP*YM

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Macromolecule #3: 6-O-phosphono-alpha-D-glucopyranose

MacromoleculeName: 6-O-phosphono-alpha-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 4 / Formula: G6P
Molecular weightTheoretical: 260.136 Da
Chemical component information

ChemComp-G6P:
6-O-phosphono-alpha-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 854608
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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