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- PDB-8vzo: Cryo-EM structure of FLVCR2 in the outward-facing state with chol... -

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Basic information

Entry
Database: PDB / ID: 8vzo
TitleCryo-EM structure of FLVCR2 in the outward-facing state with choline bound
Components
  • Fab FLV23 heavy chain
  • Fab FLV23 light chain
  • Feline leukemia virus subgroup C cellular receptor 2
KeywordsTRANSPORT PROTEIN / Choline transporter / blood-brain barrier / membrane protein / MFS fold
Function / homology
Function and homology information


heme transmembrane transporter activity / mitochondrial membrane / heme binding / endoplasmic reticulum membrane
Similarity search - Function
: / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
CHOLINE ION / Feline leukemia virus subgroup C cellular receptor 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsCater, R.J. / Mancia, F.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS129105-01 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL166866-01 United States
CitationJournal: Nature / Year: 2024
Title: Structural and molecular basis of choline uptake into the brain by FLVCR2.
Authors: Rosemary J Cater / Dibyanti Mukherjee / Eva Gil-Iturbe / Satchal K Erramilli / Ting Chen / Katie Koo / Nicolás Santander / Andrew Reckers / Brian Kloss / Tomasz Gawda / Brendon C Choy / ...Authors: Rosemary J Cater / Dibyanti Mukherjee / Eva Gil-Iturbe / Satchal K Erramilli / Ting Chen / Katie Koo / Nicolás Santander / Andrew Reckers / Brian Kloss / Tomasz Gawda / Brendon C Choy / Zhening Zhang / Aditya Katewa / Amara Larpthaveesarp / Eric J Huang / Scott W J Mooney / Oliver B Clarke / Sook Wah Yee / Kathleen M Giacomini / Anthony A Kossiakoff / Matthias Quick / Thomas Arnold / Filippo Mancia /
Abstract: Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for ...Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.
History
DepositionFeb 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Feline leukemia virus subgroup C cellular receptor 2
B: Fab FLV23 heavy chain
D: Fab FLV23 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)109,3384
Polymers109,2343
Non-polymers1041
Water0
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: Protein Feline leukemia virus subgroup C cellular receptor 2


Mass: 60103.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Flvcr2, Mfsd7c / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GNTI- / References: UniProt: A0A0R4J0E9
#2: Antibody Fab FLV23 heavy chain


Mass: 25772.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab FLV23 light chain


Mass: 23357.916 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-CHT / CHOLINE ION / Choline


Mass: 104.171 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H14NO / 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: FLVCR2 in the outward-facing state complexed with choline and Fab FLV23
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 GNTI-
Buffer solutionpH: 7
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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 58.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
7Cootmodel fitting
9Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 433845 / Symmetry type: POINT

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