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- EMDB-18667: Structure of human SPNS2 in LMNG -

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Basic information

Entry
Database: EMDB / ID: EMD-18667
TitleStructure of human SPNS2 in LMNG
Map datacryoSPARC autosharpened map from masked local refinement using for model refinement
Sample
  • Complex: Complex of SPNS2 with nanobody D12
    • Protein or peptide: Sphingosine-1-phosphate transporter SPNS2
    • Protein or peptide: Nanobody D12
KeywordsSLC TRANSPORTER / MEMBRANE PROTEIN / S1P / EXPORTER / LIPID TRANSPORT
Function / homology
Function and homology information


regulation of eye pigmentation / regulation of humoral immune response / regulation of T cell migration / sphingolipid transporter activity / lymphocyte migration / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / sphingosine-1-phosphate receptor signaling pathway / lipid transport / T cell homeostasis ...regulation of eye pigmentation / regulation of humoral immune response / regulation of T cell migration / sphingolipid transporter activity / lymphocyte migration / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / sphingosine-1-phosphate receptor signaling pathway / lipid transport / T cell homeostasis / B cell homeostasis / transmembrane transporter activity / lymph node development / sensory perception of sound / bone development / endosome membrane / membrane / plasma membrane
Similarity search - Function
Protein spinster-like / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Sphingosine-1-phosphate transporter SPNS2
Similarity search - Component
Biological speciesHomo sapiens (human) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsLi HZ / Pike ACW / McKinley G / Mukhopadhyay SMM / Moreau C / Scacioc A / Abrusci P / Borkowska O / Chalk R / Stefanic S ...Li HZ / Pike ACW / McKinley G / Mukhopadhyay SMM / Moreau C / Scacioc A / Abrusci P / Borkowska O / Chalk R / Stefanic S / Burgess-Brown N / Duerr KL / Sauer DB
Funding support Switzerland, United Kingdom, 2 items
OrganizationGrant numberCountry
Innovative Medicines Initiative777372 Switzerland
Wellcome Trust106169/Z/14/Z United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2.
Authors: Huanyu Z Li / Ashley C W Pike / Yung-Ning Chang / Dheeraj Prakaash / Zuzana Gelova / Josefina Stanka / Christophe Moreau / Hannah C Scott / Frank Wunder / Gernot Wolf / Andreea Scacioc / ...Authors: Huanyu Z Li / Ashley C W Pike / Yung-Ning Chang / Dheeraj Prakaash / Zuzana Gelova / Josefina Stanka / Christophe Moreau / Hannah C Scott / Frank Wunder / Gernot Wolf / Andreea Scacioc / Gavin McKinley / Helena Batoulis / Shubhashish Mukhopadhyay / Andrea Garofoli / Adán Pinto-Fernández / Benedikt M Kessler / Nicola A Burgess-Brown / Saša Štefanić / Tabea Wiedmer / Katharina L Dürr / Vera Puetter / Alexander Ehrmann / Syma Khalid / Alvaro Ingles-Prieto / Giulio Superti-Furga / David B Sauer /
Abstract: Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell ...Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamics simulations to probe SPNS2's substrate binding and transport. These results reveal the transporter's binding mode to its native substrate S1P, the therapeutic FTY720-P, and the reported SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein's mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein's export activity and thereby lead to hearing loss.
History
DepositionOct 17, 2023-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18667.png

Downloads & links

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Map

FileDownload / File: emd_18667.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoSPARC autosharpened map from masked local refinement using for model refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 212.736 Å		0.83 Å/pix.
x 256 pix.
= 212.736 Å		0.83 Å/pix.
x 256 pix.
= 212.736 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.831 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.67923427 - 0.98010975
Average (Standard dev.)0.000008471919 (±0.023934484)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.736 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18667_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened fullmap from local refinement

Fileemd_18667_additional_1.map
AnnotationUnsharpened fullmap from local refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Final half-map 1

Fileemd_18667_half_map_1.map
AnnotationFinal half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Final half-map 2

Fileemd_18667_half_map_2.map
AnnotationFinal half-map 2
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 SPNS2 with nanobody D12

EntireName: Complex of SPNS2 with nanobody D12
Components
  • Complex: Complex of SPNS2 with nanobody D12
    • Protein or peptide: Sphingosine-1-phosphate transporter SPNS2
    • Protein or peptide: Nanobody D12

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Supramolecule #1: Complex of SPNS2 with nanobody D12

SupramoleculeName: Complex of SPNS2 with nanobody D12 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 74.53 KDa

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Macromolecule #1: Sphingosine-1-phosphate transporter SPNS2

MacromoleculeName: Sphingosine-1-phosphate transporter SPNS2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.962211 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MMCLECASAA AGGAEEEEAD AERRRRRRGA QRGAGGSGCC GARGAGGAGV SAAGDEVQTL SGSVRRAPTG PPGTPGTPGC AATAKGPGA QQPKPASLGR GRGAAAAILS LGNVLNYLDR YTVAGVLLDI QQHFGVKDRG AGLLQSVFIC SFMVAAPIFG Y LGDRFNRK ...String:
MMCLECASAA AGGAEEEEAD AERRRRRRGA QRGAGGSGCC GARGAGGAGV SAAGDEVQTL SGSVRRAPTG PPGTPGTPGC AATAKGPGA QQPKPASLGR GRGAAAAILS LGNVLNYLDR YTVAGVLLDI QQHFGVKDRG AGLLQSVFIC SFMVAAPIFG Y LGDRFNRK VILSCGIFFW SAVTFSSSFI PQQYFWLLVL SRGLVGIGEA SYSTIAPTII GDLFTKNTRT LMLSVFYFAI PL GSGLGYI TGSSVKQAAG DWHWALRVSP VLGMITGTLI LILVPATKRG HADQLGDQLK ARTSWLRDMK ALIRNRSYVF SSL ATSAVS FATGALGMWI PLYLHRAQVV QKTAETCNSP PCGAKDSLIF GAITCFTGFL GVVTGAGATR WCRLKTQRAD PLVC AVGML GSAIFICLIF VAAKSSIVGA YICIFVGETL LFSNWAITAD ILMYVVIPTR RATAVALQSF TSHLLGDAGS PYLIG FISD LIRQSTKDSP LWEFLSLGYA LMLCPFVVVL GGMFFLATAL FFVSDRARAE QQVNQLAMPP ASVKVAENLY FQ

UniProtKB: Sphingosine-1-phosphate transporter SPNS2

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Macromolecule #2: Nanobody D12

MacromoleculeName: Nanobody D12 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 15.674475 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGRLLS WYDMAWFRQA PGKEREFVAA VTSTGAGTHY VDSVKGRFTI SRVNAKNTMY LQMNSLKPE DTAVYYCAAA NTRLTALSLR TTTGSWAYWG KGTPVTVSSA DYKDDDDKHH HHHH

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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

Concentration6 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
150.0 mMSodium chloride
0.002 %Lauryl Maltose Neopentyl Glyco (LMNG)

Details: 20mM HEPES pH 7.5, 150mM NaCl, 0.002% LMNG
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSEC purified

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailseBIC Krios II
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8680 / Average exposure time: 2.0 sec. / Average electron dose: 20.71 e/Å2 / Details: eBIC Diamond Krios II
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3376229
Startup modelType of model: OTHER / Details: cryoSPARC Ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 308935
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.1)
Details: Mulitple rounds of ab-intio (two classes) followed by heterogeneous refinement to converge on final set of particles
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: OTHER
Output model

PDB-8qv5:
Structure of human SPNS2 in LMNG

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