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- EMDB-19753: Cryo-EM structure of hydroxychloroquine-bound human SLC19A3 in in... -

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Basic information

Entry
Database: EMDB / ID: EMD-19753
TitleCryo-EM structure of hydroxychloroquine-bound human SLC19A3 in inward-open state
Map data
Sample
  • Complex: SLC19A3:Nb3.7:hydroxychloroquine
    • Complex: SLC19A3
      • Protein or peptide: Thiamine transporter 2
    • Complex: Nb3.3
      • Protein or peptide: Nb3.3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: Hydroxychloroquine
KeywordsSLC19A3 / vitamin transporter / thiamine transporter / MFS fold / nanobody complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


pyridoxine transport / thiamine-containing compound metabolic process / Vitamin B1 (thiamin) metabolism / thiamine transmembrane transport / thiamine transmembrane transporter activity / thiamine transport / thiamine diphosphate biosynthetic process / transmembrane transport / membrane / plasma membrane
Similarity search - Function
Thiamine transporter 2 / Reduced folate carrier / Reduced folate carrier / MFS transporter superfamily
Similarity search - Domain/homology
Thiamine transporter 2
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsGabriel F / Loew C
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)10251European Union
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of thiamine transport and drug recognition by SLC19A3.
Authors: Florian Gabriel / Lea Spriestersbach / Antonia Fuhrmann / Katharina E J Jungnickel / Siavash Mostafavi / Els Pardon / Jan Steyaert / Christian Löw /
Abstract: Thiamine (vitamin B) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell ...Thiamine (vitamin B) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane, which is mainly mediated by the two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters lead to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report seven cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enable us to capture different states of SLC19A3's transport cycle, revealing the molecular details of thiamine recognition and transport. We identify seven previously unknown drug interactions of SLC19A3 and present structures of the transporter in complex with the inhibitors fedratinib, amprolium and hydroxychloroquine. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.
History
DepositionFeb 26, 2024-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19753.png

Downloads & links

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Map

FileDownload / File: emd_19753.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.57 Å/pix.
x 512 pix.
= 292.864 Å		0.57 Å/pix.
x 512 pix.
= 292.864 Å		0.57 Å/pix.
x 512 pix.
= 292.864 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.572 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.85
Minimum - Maximum-4.6462355 - 6.592825
Average (Standard dev.)-0.00034757634 (±0.072925396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 292.864 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19753_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_19753_half_map_1.map
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Half map: #2

Fileemd_19753_half_map_2.map
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Sample components

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Entire : SLC19A3:Nb3.7:hydroxychloroquine

EntireName: SLC19A3:Nb3.7:hydroxychloroquine
Components
  • Complex: SLC19A3:Nb3.7:hydroxychloroquine
    • Complex: SLC19A3
      • Protein or peptide: Thiamine transporter 2
    • Complex: Nb3.3
      • Protein or peptide: Nb3.3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: Hydroxychloroquine

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Supramolecule #1: SLC19A3:Nb3.7:hydroxychloroquine

SupramoleculeName: SLC19A3:Nb3.7:hydroxychloroquine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: SLC19A3

SupramoleculeName: SLC19A3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nb3.3

SupramoleculeName: Nb3.3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Nb3.3

MacromoleculeName: Nb3.3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 15.205778 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQAGDSLRL SCAASGRTFS NYYMAWFRQA PGKEREFVAA IRLSYGSTYY ADSVRGRFTI SKDNAKNTVN LRMNSLKSE DTAIYYCAAA EDRWALAVRT ATTYNYWGQG TQVTVSSHHH HHHEPEA

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Macromolecule #2: Thiamine transporter 2

MacromoleculeName: Thiamine transporter 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 59.687609 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDCYRTSLSS SWIYPTVILC LFGFFSMMRP SEPFLIPYLS GPDKNLTSAE ITNEIFPVWT YSYLVLLLPV FVLTDYVRYK PVIILQGIS FIITWLLLLF GQGVKTMQVV EFFYGMVTAA EVAYYAYIYS VVSPEHYQRV SGYCRSVTLA AYTAGSVLAQ L LVSLANMS ...String:
MDCYRTSLSS SWIYPTVILC LFGFFSMMRP SEPFLIPYLS GPDKNLTSAE ITNEIFPVWT YSYLVLLLPV FVLTDYVRYK PVIILQGIS FIITWLLLLF GQGVKTMQVV EFFYGMVTAA EVAYYAYIYS VVSPEHYQRV SGYCRSVTLA AYTAGSVLAQ L LVSLANMS YFYLNVISLA SVSVAFLFSL FLPMPKKSMF FHAKPSREIK KSSSVNPVLE ETHEGEAPGC EEQKPTSEIL ST SGKLNKG QLNSLKPSNV TVDVFVQWFQ DLKECYSSKR LFYWSLWWAF ATAGFNQVLN YVQILWDYKA PSQDSSIYNG AVE AIATFG GAVAAFAVGY VKVNWDLLGE LALVVFSVVN AGSLFLMHYT ANIWACYAGY LIFKSSYMLL ITIAVFQIAV NLNV ERYAL VFGINTFIAL VIQTIMTVIV VDQRGLNLPV SIQFLVYGSY FAVIAGIFLM RSMYITYSTK SQKDVQSPAP SENPD VSHP EEESNIIMST KLLEVLFQGP SSGWSHPQFE KGGGSGGGSG GSAWSHPQFE K

UniProtKB: Thiamine transporter 2

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: Hydroxychloroquine

MacromoleculeName: Hydroxychloroquine / type: ligand / ID: 4 / Number of copies: 1 / Formula: A1H5D
Molecular weightTheoretical: 335.872 Da

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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

BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
150.0 mMsodium chlorideNaCl
0.002 %LMNG
0.0002 %CHS
0.25 mMhydroxychloroqine
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 9492 / Average exposure time: 2.1 sec. / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
Sample stageCooling holder cryogen: NITROGEN
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: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 249857
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8s5z:
Cryo-EM structure of hydroxychloroquine-bound human SLC19A3 in inward-open state

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