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- PDB-8pd9: cAMP-bound SpSLC9C1 in lipid nanodiscs, protomer state 1 -

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Basic information

Entry
Database: PDB / ID: 8pd9
TitlecAMP-bound SpSLC9C1 in lipid nanodiscs, protomer state 1
ComponentsSperm-specific sodium proton exchanger
KeywordsMEMBRANE PROTEIN / SLC9 / NHE / sperm-specific
Function / homology
Function and homology information


sperm head / potassium:proton antiporter activity / sodium:proton antiporter activity / sodium ion import across plasma membrane / cGMP binding / sperm flagellum / single fertilization / cAMP binding / potassium ion transmembrane transport / regulation of intracellular pH ...sperm head / potassium:proton antiporter activity / sodium:proton antiporter activity / sodium ion import across plasma membrane / cGMP binding / sperm flagellum / single fertilization / cAMP binding / potassium ion transmembrane transport / regulation of intracellular pH / protein homodimerization activity / plasma membrane
Similarity search - Function
Cation/H+ exchanger, CPA1 family / Cation/H+ exchanger / Sodium/hydrogen exchanger, transmembrane / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Voltage-dependent channel domain superfamily / RmlC-like jelly roll fold
Similarity search - Domain/homology
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / Sperm-specific sodium:proton exchanger
Similarity search - Component
Biological speciesStrongylocentrotus purpuratus (purple sea urchin)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKalienkova, V. / Peter, M. / Rheinberger, J. / Paulino, C.
Funding support Netherlands, Switzerland, 4items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Swiss National Science FoundationP500PB_203053 Switzerland
Swiss National Science FoundationP2ZHP3_187679 Switzerland
CitationJournal: Nature / Year: 2023
Title: Structures of a sperm-specific solute carrier gated by voltage and cAMP.
Authors: Valeria Kalienkova / Martin F Peter / Jan Rheinberger / Cristina Paulino /
Abstract: The newly characterized sperm-specific Na/H exchanger stands out by its unique tripartite domain composition. It unites a classical solute carrier unit with regulatory domains usually found in ion ...The newly characterized sperm-specific Na/H exchanger stands out by its unique tripartite domain composition. It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain, which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals, our structure represents a potential new platform for the development of new on-demand contraceptives.
History
DepositionJun 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sperm-specific sodium proton exchanger
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,9542
Polymers147,6241
Non-polymers3291
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 Sperm-specific sodium proton exchanger


Mass: 147624.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Strongylocentrotus purpuratus (purple sea urchin)
Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: A3RL54
#2: Chemical ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.206 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H12N5O6P / 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: SpSLC9C1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Strongylocentrotus purpuratus (purple sea urchin)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293S GnTI-
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMNaCl1
32.9 mMfluorinated fos-choline 81
SpecimenConc.: 11.46 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 7 / Num. of real images: 25363
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9RELION3.1.0initial Euler assignment
10RELION3.1.0final Euler assignment
11RELION3.1.0classification
12RELION3.1.03D reconstruction
19ISOLDEmodel refinement
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6921839
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 357595 / Symmetry type: POINT

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