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- PDB-8hik: The TPP-bound BRIL-SLC19A1/Fab/Nb ternary complex -

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Basic information

Entry
Database: PDB / ID: 8hik
TitleThe TPP-bound BRIL-SLC19A1/Fab/Nb ternary complex
Components
  • Anti-BRIL Fab heavy chain
  • Anti-BRIL Fab light chain
  • Anti-Fab nanobody
  • BRIL-SLC19A1 chimera
KeywordsTRANSPORT PROTEIN / SLC19A1 / RFC / transporter / folates / 5-MTHF / TPP
Function / homology
Function and homology information


folic acid transmembrane transporter activity / folate:monoatomic anion antiporter activity / methotrexate transport / folate transmembrane transport / methotrexate transmembrane transporter activity / folic acid transport / folate import across plasma membrane / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / organic anion transport ...folic acid transmembrane transporter activity / folate:monoatomic anion antiporter activity / methotrexate transport / folate transmembrane transport / methotrexate transmembrane transporter activity / folic acid transport / folate import across plasma membrane / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / organic anion transport / 2',3'-cyclic GMP-AMP binding / xenobiotic transmembrane transport / organic anion transmembrane transporter activity / Metabolism of folate and pterines / antiporter activity / folic acid binding / folic acid metabolic process / xenobiotic transmembrane transporter activity / transport across blood-brain barrier / electron transport chain / female pregnancy / brush border membrane / basolateral plasma membrane / electron transfer activity / periplasmic space / iron ion binding / apical plasma membrane / heme binding / plasma membrane
Similarity search - Function
Reduced folate transporter SLC19A1 / Reduced folate carrier / Reduced folate carrier / MFS transporter superfamily / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562
Similarity search - Domain/homology
THIAMINE DIPHOSPHATE / Soluble cytochrome b562 / Reduced folate transporter
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsZhang, Z. / Dang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32171201 China
CitationJournal: Cell Discov / Year: 2022
Title: Molecular mechanism of substrate recognition by folate transporter SLC19A1.
Authors: Yu Dang / Dong Zhou / Xiaojuan Du / Hongtu Zhao / Chia-Hsueh Lee / Jing Yang / Yijie Wang / Changdong Qin / Zhenxi Guo / Zhe Zhang /
Abstract: Folate (vitamin B) is the coenzyme involved in one-carbon transfer biochemical reactions essential for cell survival and proliferation, with its inadequacy causing developmental defects or severe ...Folate (vitamin B) is the coenzyme involved in one-carbon transfer biochemical reactions essential for cell survival and proliferation, with its inadequacy causing developmental defects or severe diseases. Notably, mammalian cells lack the ability to de novo synthesize folate but instead rely on its intake from extracellular sources via specific transporters or receptors, among which SLC19A1 is the ubiquitously expressed one in tissues. However, the mechanism of substrate recognition by SLC19A1 remains unclear. Here we report the cryo-EM structures of human SLC19A1 and its complex with 5-methyltetrahydrofolate at 3.5-3.6 Å resolution and elucidate the critical residues for substrate recognition. In particular, we reveal that two variant residues among SLC19 subfamily members designate the specificity for folate. Moreover, we identify intracellular thiamine pyrophosphate as the favorite coupled substrate for folate transport by SLC19A1. Together, this work establishes the molecular basis of substrate recognition by this central folate transporter.
History
DepositionNov 20, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: BRIL-SLC19A1 chimera
H: Anti-BRIL Fab heavy chain
N: Anti-Fab nanobody
L: Anti-BRIL Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,5445
Polymers145,1194
Non-polymers4251
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein BRIL-SLC19A1 chimera


Mass: 75110.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, SLC19A1, FLOT1, RFC1 / Production host: Homo sapiens (human) / References: UniProt: P0ABE7, UniProt: P41440
#2: Antibody Anti-BRIL Fab heavy chain


Mass: 27759.207 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody Anti-Fab nanobody


Mass: 16673.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21 (bacteria)
#4: Antibody Anti-BRIL Fab light chain


Mass: 25575.604 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Chemical ChemComp-TPP / THIAMINE DIPHOSPHATE / Thiamine pyrophosphate


Mass: 425.314 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H19N4O7P2S / Feature type: SUBJECT OF INVESTIGATION
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: The TPP-bound BRIL-SLC19A1/Fab/Nb ternary complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
11cryoSPARCfinal Euler assignment
CTF correctionType: NONE
3D reconstructionResolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229158 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038445
ELECTRON MICROSCOPYf_angle_d0.77611494
ELECTRON MICROSCOPYf_dihedral_angle_d8.5174948
ELECTRON MICROSCOPYf_chiral_restr0.0471296
ELECTRON MICROSCOPYf_plane_restr0.0061437

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