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- PDB-8qv6: Structure of human SPNS2 in DDM -

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Basic information

Entry
Database: PDB / ID: 8qv6
TitleStructure of human SPNS2 in DDM
Components
  • Nanobody D12
  • Sphingosine-1-phosphate transporter SPNS2
KeywordsLIPID TRANSPORT / SLC TRANSPORTER / MEMBRANE PROTEIN / S1P / EXPORTER
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsLi, H.Z. / Pike, A.C.W. / McKinley, G. / Mukhopadhyay, S.M.M. / Moreau, C. / Scacioc, A. / Abrusci, P. / Borkowska, O. / Chalk, R. / Stefanic, S. ...Li, H.Z. / Pike, A.C.W. / McKinley, G. / Mukhopadhyay, S.M.M. / Moreau, C. / Scacioc, A. / Abrusci, P. / Borkowska, O. / Chalk, R. / Stefanic, S. / Burgess-Brown, N. / Duerr, K.L. / Sauer, D.B.
Funding support Switzerland, United Kingdom, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sphingosine-1-phosphate transporter SPNS2
B: Nanobody D12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,1473
Polymers74,6372
Non-polymers5111
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: Protein Sphingosine-1-phosphate transporter SPNS2


Mass: 58962.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPNS2 / Plasmid: pHTBV1.1-CTGFP-SIII-10H / Cell (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q8IVW8
#2: Antibody Nanobody D12


Mass: 15674.475 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pBXNPFLAGH / Production host: Escherichia coli (E. coli)
#3: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H46O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*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

ComponentName: Complex of SPNS2 with nanobody D12 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.07453 ° / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 20mM HEPES pH 7.5, 150mM NaCl, 0.025% DDM
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMSodium chloride1
30.025 %Dodecylmaltoside (DDM)1
SpecimenConc.: 14 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: SEC purified
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.6 sec. / Electron dose: 25.4641 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12014 / Details: eBIC Diamond Krios IV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2Topazparticle selection
3EPUimage acquisition
5cryoSPARC3.3.1CTF correction
8UCSF ChimeraXmodel fitting
9Coot0.9.8model fitting
11PHENIX1.20_4459model refinement
12cryoSPARC3.3.1initial Euler assignment
13cryoSPARC3.3.1final Euler assignment
14cryoSPARC3.3.1classification
15cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6672673
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 246225 / Algorithm: FOURIER SPACE
Details: Masked Local refinement in cryoSPARC with detergent micelle omitted
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 98.11 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00254198
ELECTRON MICROSCOPYf_angle_d0.46075720
ELECTRON MICROSCOPYf_chiral_restr0.036676
ELECTRON MICROSCOPYf_plane_restr0.003707
ELECTRON MICROSCOPYf_dihedral_angle_d9.65391391

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