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- PDB-9boi: Cryo-EM structure of human Spns1 in complex with LPC (18:1) -

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Basic information

Entry
Database: PDB / ID: 9boi
TitleCryo-EM structure of human Spns1 in complex with LPC (18:1)
ComponentsmVenus,Maltose/maltodextrin-binding periplasmic protein,Protein spinster homolog 1,Designed ankyrin repeat protein (DARPin)
KeywordsMEMBRANE PROTEIN / lysosome / major facilitator superfamily / lysophospholipid transporter / lysophosphatidylcholine
Function / homology
Function and homology information


lysophospholipid transport / regulation of lysosomal lumen pH / phospholipid efflux / detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / transmembrane transporter activity / maltose binding / maltose transport ...lysophospholipid transport / regulation of lysosomal lumen pH / phospholipid efflux / detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / bioluminescence / cell chemotaxis / generation of precursor metabolites and energy / outer membrane-bounded periplasmic space / periplasmic space / mitochondrial inner membrane / lysosomal membrane / DNA damage response / membrane
Similarity search - Function
Protein spinster-like / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / MFS transporter superfamily / Bacterial extracellular solute-binding protein ...Protein spinster-like / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / MFS transporter superfamily / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Chem-42H / Maltose/maltodextrin-binding periplasmic protein / Green fluorescent protein / Protein spinster homolog 1
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Escherichia coli (E. coli)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsChen, H. / Li, X.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01 HL160487 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM135343 United States
Welch FoundationI-1957 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Molecular basis of Spns1-mediated lysophospholipid transport from the lysosome.
Authors: Hongwen Chen / Hoa T T Ha / Nadia Elghobashi-Meinhardt / Nhung A Le / Philip Schmiege / Long N Nguyen / Xiaochun Li /
Abstract: Spns1 mediates the rate-limiting efflux of lysophospholipids from the lysosome to the cytosol. Deficiency of Spns1 is associated with embryonic senescence, as well as liver and skeletal muscle ...Spns1 mediates the rate-limiting efflux of lysophospholipids from the lysosome to the cytosol. Deficiency of Spns1 is associated with embryonic senescence, as well as liver and skeletal muscle atrophy in animal models. However, the mechanisms by which Spns1 transports lysophospholipid and proton sensing remain unclear. Here, we present a cryogenic electron microscopy structure of human Spns1 in lysophosphatidylcholine (LPC)-bound lumen-facing conformation. Notably, LPC snugly binds within the luminal-open cavity, where the molecular dynamics simulations reveal that LPC presents a propensity to enter between transmembrane-helices (TM) 5 and 8. Structural comparisons and cell-based transport assays uncover several pivotal residues at TM 5/8 that orchestrate the transport cycle, which are unique to Spns1. Furthermore, we identify a five-residue network that is crucial for proton-sensing by Spns1. Transference of these network residues to Spns2, a sphingosine-1-phosphate uniporter, causes the chimeric Spns2 to be low pH dependent. Our results reveal molecular insights into lysosomal LPC transport and the proton-sensing mechanism by Spns1.
History
DepositionMay 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: mVenus,Maltose/maltodextrin-binding periplasmic protein,Protein spinster homolog 1,Designed ankyrin repeat protein (DARPin)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,8222
Polymers139,3001
Non-polymers5231
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 mVenus,Maltose/maltodextrin-binding periplasmic protein,Protein spinster homolog 1,Designed ankyrin repeat protein (DARPin) / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / HSpin1 / SPNS1 / Spinster-like protein 1


Mass: 139299.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: GFP, malE, SPNS1, SPIN1, PP20300 / Production host: Homo sapiens (human)
References: UniProt: P42212, UniProt: P0AEX9, UniProt: Q9H2V7
#2: Chemical ChemComp-42H / (4R,7R,18Z)-4,7-dihydroxy-N,N,N-trimethyl-10-oxo-3,5,9-trioxa-4-phosphaheptacos-18-en-1-aminium 4-oxide


Mass: 522.675 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H53NO7P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: human Spns1 purified in the presence of extra LPC addition
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTI-
Buffer solutionpH: 5.5
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 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 defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF Chimera1.15model fitting
9PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 614781 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0043332
ELECTRON MICROSCOPYf_angle_d0.7174534
ELECTRON MICROSCOPYf_dihedral_angle_d7.8621925
ELECTRON MICROSCOPYf_chiral_restr0.047532
ELECTRON MICROSCOPYf_plane_restr0.006561

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