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- PDB-8s61: Cryo-EM structure of thiamine-bound human SLC19A3 in inward-open state -

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Basic information

Entry
Database: PDB / ID: 8s61
TitleCryo-EM structure of thiamine-bound human SLC19A3 in inward-open state
Components
  • Nb3.7
  • Thiamine transporter 2
KeywordsMEMBRANE PROTEIN / SLC19A3 / vitamin transporter / thiamine transporter / MFS fold / nanobody complex
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
Chem-VIB / Thiamine transporter 2
Similarity search - Component
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsGabriel, F. / Loew, C.
Funding supportEuropean Union, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 2.0Oct 9, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / em_imaging / entity / entity_poly / entity_poly_seq / entity_src_gen / pdbx_entry_details / pdbx_modification_feature / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / refine / refine_ls_restr / refine_ls_shell / struct_conf / struct_conn / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet_range
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_seq_id / _atom_site.label_seq_id / _atom_site.occupancy / _em_admin.last_update / _em_imaging.microscope_model / _entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_end_seq_num / _pdbx_entry_details.has_protein_modification / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _struct_conf.beg_label_seq_id / _struct_conf.end_label_seq_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_label_seq_id / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.seq_align_end / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_label_seq_id
Description: Atomic clashes / Provider: author / Type: Coordinate replacement
Revision 2.1Oct 16, 2024Group: Data collection / Database references / Category: 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 / _em_admin.last_update
Revision 2.2Nov 6, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 2.3Dec 4, 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
B: Nb3.7
A: Thiamine transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,0703
Polymers63,8042
Non-polymers2651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Antibody Nb3.7


Mass: 13296.790 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#2: Protein Thiamine transporter 2 / ThTr-2 / ThTr2 / Solute carrier family 19 member 3


Mass: 50507.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC19A3 / Production host: Homo sapiens (human) / References: UniProt: Q9BZV2
#3: Chemical ChemComp-VIB / 3-(4-AMINO-2-METHYL-PYRIMIDIN-5-YLMETHYL)-5-(2-HYDROXY-ETHYL)-4-METHYL-THIAZOL-3-IUM / THIAMIN / VITAMIN B1


Mass: 265.355 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H17N4OS / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
Has ligand of interestY
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1SLC19A3:Nb3.7:thiamineCOMPLEX#10RECOMBINANT
2SLC19A3COMPLEX#21RECOMBINANT
3Nb3.7COMPLEX#11RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
2150 mMsodium chlorideNaCl1
30.002 %LMNG1
40.0002 %CHS1
50.5 mMthiamine1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5308
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 501165 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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