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- EMDB-16345: Structure of SLC40/ferroportin in complex with vamifeport and syn... -

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Basic information

Entry
Database: EMDB / ID: EMD-16345
TitleStructure of SLC40/ferroportin in complex with vamifeport and synthetic nanobody Sy3 in occluded conformation
Map dataRefined and sharpened map used for model building and refinement
Sample
  • Complex: Complex of ferroportin with synthetic nanobody and inhibitor
    • Protein or peptide: Solute carrier family 40 member 1
    • Protein or peptide: Sybody 3
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE
  • Ligand: 2-[2-[2-(1~{H}-benzimidazol-2-yl)ethylamino]ethyl]-~{N}-[(3-fluoranylpyridin-2-yl)methyl]-1,3-oxazole-4-carboxamide
Function / homology
Function and homology information


spleen trabecula formation / iron ion export across plasma membrane / Defective SLC40A1 causes hemochromatosis 4 (HFE4) (duodenum) / Defective SLC40A1 causes hemochromatosis 4 (HFE4) (macrophages) / Defective CP causes aceruloplasminemia (ACERULOP) / Metal ion SLC transporters / iron ion transmembrane transport / lymphocyte homeostasis / iron ion transmembrane transporter activity / ferrous iron transmembrane transporter activity ...spleen trabecula formation / iron ion export across plasma membrane / Defective SLC40A1 causes hemochromatosis 4 (HFE4) (duodenum) / Defective SLC40A1 causes hemochromatosis 4 (HFE4) (macrophages) / Defective CP causes aceruloplasminemia (ACERULOP) / Metal ion SLC transporters / iron ion transmembrane transport / lymphocyte homeostasis / iron ion transmembrane transporter activity / ferrous iron transmembrane transporter activity / endothelium development / peptide hormone binding / establishment of localization in cell / Iron uptake and transport / multicellular organismal-level iron ion homeostasis / synaptic vesicle / basolateral plasma membrane / intracellular iron ion homeostasis / transcription by RNA polymerase II / apoptotic process / negative regulation of apoptotic process / positive regulation of transcription by RNA polymerase II / nucleoplasm / membrane / identical protein binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ferroportin-1 / Ferroportin1 (FPN1) / MFS transporter superfamily
Similarity search - Domain/homology
Solute carrier family 40 member 1
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsLehmann EF / Liziczai M / Drozdzyk K / Dutzler R / Manatschal C
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Elife / Year: 2023
Title: Structures of ferroportin in complex with its specific inhibitor vamifeport.
Authors: Elena Farah Lehmann / Márton Liziczai / Katarzyna Drożdżyk / Patrick Altermatt / Cassiano Langini / Vania Manolova / Hanna Sundstrom / Franz Dürrenberger / Raimund Dutzler / Cristina Manatschal /
Abstract: A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe transport and induces ...A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter.
History
DepositionDec 15, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateMay 24, 2023-
Current statusMay 24, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16345.png

Downloads & links

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Map

FileDownload / File: emd_16345.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined and sharpened map used for model building and refinement
Voxel sizeX=Y=Z: 0.651 Å
Density
Contour LevelBy AUTHOR: 0.264
Minimum - Maximum-0.9984431 - 1.4742655
Average (Standard dev.)0.0004952776 (±0.02787383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 234.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16345_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: One of the half maps (A)

Fileemd_16345_half_map_1.map
AnnotationOne of the half maps (A)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Second half map (B)

Fileemd_16345_half_map_2.map
AnnotationSecond half map (B)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of ferroportin with synthetic nanobody and inhibitor

EntireName: Complex of ferroportin with synthetic nanobody and inhibitor
Components
  • Complex: Complex of ferroportin with synthetic nanobody and inhibitor
    • Protein or peptide: Solute carrier family 40 member 1
    • Protein or peptide: Sybody 3
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE
  • Ligand: 2-[2-[2-(1~{H}-benzimidazol-2-yl)ethylamino]ethyl]-~{N}-[(3-fluoranylpyridin-2-yl)methyl]-1,3-oxazole-4-carboxamide

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Supramolecule #1: Complex of ferroportin with synthetic nanobody and inhibitor

SupramoleculeName: Complex of ferroportin with synthetic nanobody and inhibitor
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Details: Ferroportin was expressed in human derived HEK cells, whereas the synthetic nanobody was expressed in bacterial cell culture. The two were purified separately and mixed shortly before vitrification
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Solute carrier family 40 member 1

MacromoleculeName: Solute carrier family 40 member 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.536848 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSTRAGDHNR QRGCCGSLAD YLTSAKFLLY LGHSLSTWGD RMWHFAVSVF LVELYGNSLL LTAVYGLVVA GSVLVLGAII GDWVDKNAR LKVAQTSLVV QNVSVILCGI ILMMVFLHKH ELLTMYHGWV LTSCYILIIT IANIANLAST ATAITIQRDW I VVVAGEDR ...String:
MSTRAGDHNR QRGCCGSLAD YLTSAKFLLY LGHSLSTWGD RMWHFAVSVF LVELYGNSLL LTAVYGLVVA GSVLVLGAII GDWVDKNAR LKVAQTSLVV QNVSVILCGI ILMMVFLHKH ELLTMYHGWV LTSCYILIIT IANIANLAST ATAITIQRDW I VVVAGEDR SKLANMNATI RRIDQLTNIL APMAVGQIMT FGSPVIGCGF ISGWNLVSMC VEYVLLWKVY QKTPALAVKA GL KEEETEL KQLNLHKDTE PKPLEGTHLM GVKDSNIHEL EHEQEPTCAS QMAEPFRTFR DGWVSYYNQP VFLAGMGLAF LYM TVLGFD CITTGYAYTQ GLSGSILSIL MGASAITGIM GTVAFTWLRR KCGLVRTGLI SGLAQLSCLI LCVISVFMPG SPLD LSVSP FEDIRSRFIQ GESITPTKIP EITTEIYMSN GSNSANIVPE TSPESVPIIS VSLLFAGVIA ARIGLWSFDL TVTQL LQEN VIESERGIIN GVQNSMNYLL DLLHFIMVIL APNPEAFGLL VLISVSFVAM GHIMYFRFAQ NTLGNKLFAC GPDAKE VRK ENQANTSVVA LEVLFQG

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Macromolecule #2: Sybody 3

MacromoleculeName: Sybody 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.343104 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGFPVA WNEMRWYRQA PGKEREWVAA IASIGVTTYY ADSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCNVK DYGMAFWYYD YWGQGTQVTV SAGRAGEQKL ISEEDLNSAV DHHHHHH

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Macromolecule #3: DIUNDECYL PHOSPHATIDYL CHOLINE

MacromoleculeName: DIUNDECYL PHOSPHATIDYL CHOLINE / type: ligand / ID: 3 / Number of copies: 1 / Formula: PLC
Molecular weightTheoretical: 622.834 Da
Chemical component information

ChemComp-PLC:
DIUNDECYL PHOSPHATIDYL CHOLINE / phospholipid*YM

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Macromolecule #4: 2-[2-[2-(1~{H}-benzimidazol-2-yl)ethylamino]ethyl]-~{N}-[(3-fluor...

MacromoleculeName: 2-[2-[2-(1~{H}-benzimidazol-2-yl)ethylamino]ethyl]-~{N}-[(3-fluoranylpyridin-2-yl)methyl]-1,3-oxazole-4-carboxamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: SZU
Molecular weightTheoretical: 408.429 Da
Chemical component information

ChemComp-SZU:
2-[2-[2-(1~{H}-benzimidazol-2-yl)ethylamino]ethyl]-~{N}-[(3-fluoranylpyridin-2-yl)methyl]-1,3-oxazole-4-carboxamide

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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
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 70.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 320701
FSC plot (resolution estimation)

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