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- EMDB-17971: Cryo-EM structure of horse Nhe9 bound to PI(3,5)P2 -

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Basic information

Entry
Database: EMDB / ID: EMD-17971
TitleCryo-EM structure of horse Nhe9 bound to PI(3,5)P2
Map data
Sample
  • Complex: Dimeric Horse Nhe9 with PI(3,5)P2
    • Protein or peptide: Sodium/hydrogen exchanger 9
  • Ligand: (2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl dioctanoate
KeywordsNa+/H+ exchanger / membrane protein / SLC9A9 / ion transporter / PI(3 / 5)P2 / TRANSPORT PROTEIN
Function / homology
Function and homology information


phagosome maturation / potassium:proton antiporter activity / sodium:proton antiporter activity / early phagosome / sodium ion import across plasma membrane / potassium ion transmembrane transport / sodium ion transmembrane transport / proton transmembrane transport / regulation of intracellular pH / recycling endosome ...phagosome maturation / potassium:proton antiporter activity / sodium:proton antiporter activity / early phagosome / sodium ion import across plasma membrane / potassium ion transmembrane transport / sodium ion transmembrane transport / proton transmembrane transport / regulation of intracellular pH / recycling endosome / recycling endosome membrane / phagocytic vesicle membrane / late endosome membrane / early endosome membrane / defense response to bacterium / protein homodimerization activity / plasma membrane
Similarity search - Function
Sodium/hydrogen exchanger 6/7/9 / Na+/H+ exchanger / Cation/H+ exchanger, CPA1 family / Cation/H+ exchanger / Sodium/hydrogen exchanger, transmembrane
Similarity search - Domain/homology
Sodium/hydrogen exchanger 9
Similarity search - Component
Biological speciesEquus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsKokane S / Meier P / Gulati A / Delemotte L / Drew D
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)CoG-820187European Union
CitationJournal: Nat Commun / Year: 2025
Title: PIP-mediated oligomerization of the endosomal sodium/proton exchanger NHE9.
Authors: Surabhi Kokane / Ashutosh Gulati / Pascal F Meier / Rei Matsuoka / Tanadet Pipatpolkai / Giuseppe Albano / Tin Manh Ho / Lucie Delemotte / Daniel Fuster / David Drew /
Abstract: The strict exchange of Na for H ions across cell membranes is a reaction carried out in almost every cell. Na/H exchangers that perform this task are physiological homodimers, and whilst the ion ...The strict exchange of Na for H ions across cell membranes is a reaction carried out in almost every cell. Na/H exchangers that perform this task are physiological homodimers, and whilst the ion transporting domain is highly conserved, their dimerization differs. The Na/H exchanger NhaA from Escherichia coli has a weak dimerization interface mediated by a β-hairpin domain and with dimer retention dependent on cardiolipin. Similarly, organellar Na/H exchangers NHE6, NHE7 and NHE9 also contain β-hairpin domains and recent analysis of Equus caballus NHE9 indicated PIP lipids could bind at the dimer interface. However, structural validation of the predicted lipid-mediated oligomerization has been lacking. Here, we report cryo-EM structures of E. coli NhaA and E. caballus NHE9 in complex with cardiolipin and phosphatidylinositol-3,5-bisphosphate PI(3,5)P lipids binding at their respective dimer interfaces. We further show how the endosomal specific PI(3,5)P lipid stabilizes the NHE9 homodimer and enhances transport activity. Indeed, we show that NHE9 is active in endosomes, but not at the plasma membrane where the PI(3,5)P lipid is absent. Thus, specific lipids can regulate Na/H exchange activity by stabilizing dimerization in response to either cell specific cues or upon trafficking to their correct membrane location.
History
DepositionJul 18, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17971.png

Downloads & links

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Map

FileDownload / File: emd_17971.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 300 pix.
= 292.38 Å		0.97 Å/pix.
x 300 pix.
= 292.38 Å		0.97 Å/pix.
x 300 pix.
= 292.38 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9746 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.14286806 - 0.33065563
Average (Standard dev.)-0.000017042035 (±0.011287188)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 292.38 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17971_msk_1.map
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Half map: #2

Fileemd_17971_half_map_1.map
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Half map: #1

Fileemd_17971_half_map_2.map
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Sample components

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Entire : Dimeric Horse Nhe9 with PI(3,5)P2

EntireName: Dimeric Horse Nhe9 with PI(3,5)P2
Components
  • Complex: Dimeric Horse Nhe9 with PI(3,5)P2
    • Protein or peptide: Sodium/hydrogen exchanger 9
  • Ligand: (2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl dioctanoate

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Supramolecule #1: Dimeric Horse Nhe9 with PI(3,5)P2

SupramoleculeName: Dimeric Horse Nhe9 with PI(3,5)P2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Equus caballus (horse)
Molecular weightTheoretical: 64 KDa

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Macromolecule #1: Sodium/hydrogen exchanger 9

MacromoleculeName: Sodium/hydrogen exchanger 9 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Equus caballus (horse)
Molecular weightTheoretical: 64.856055 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSEKDEYQFQ HQGAVELLVF NFLLILTILT IWLFKNHRFR FLHETGGAMV YGLIMGLILR YATAPTDIES GTVYDCGKLA FSPSTLLIN ITDQVYEYKY KREISQHNIN PHLGNAILEK MTFDPEIFFN VLCPPIIFHA GYSLKKRHFF QNLGSILTYA F LGTAISCI ...String:
MSEKDEYQFQ HQGAVELLVF NFLLILTILT IWLFKNHRFR FLHETGGAMV YGLIMGLILR YATAPTDIES GTVYDCGKLA FSPSTLLIN ITDQVYEYKY KREISQHNIN PHLGNAILEK MTFDPEIFFN VLCPPIIFHA GYSLKKRHFF QNLGSILTYA F LGTAISCI VIGLIMYGFV KAMVYAGQLK NGDFHFTDCL FFGSLMSATD PVTVLAIFHE LHVDPDLYTL LFGESVLNDA VA IVLTYSI SIYSPKENPN AFDAAAFFQS VGNFLGIFAG SFAMGSAYAV VTALLTKFTK LCEFPMLETG LFFLLSWSAF LSA EAAGLT GIVAVLFCGV TQAHYTYNNL SLDSKMRTKQ LFEFMNFLAE NVIFCYMGLA LFTFQNHIFN ALFILGAFLA IFVA RACNI YPLSFLLNLG RKHKIPWNFQ HMMMFSGLRG ACAFALAIRD TESQPKQMMF STTLLLVFFT VWVFGGGTTP MLTWL QIRV GVDLDEDLKE RPSSHQEANN LEKSTTKTES AWLFRMWYGF DHKYLKPILT HSGPPLTTTL PEWCGPISRL LTSPQA YGE QLKEGENLYF Q

UniProtKB: Sodium/hydrogen exchanger 9

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Macromolecule #2: (2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bi...

MacromoleculeName: (2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl dioctanoate
type: ligand / ID: 2 / Number of copies: 2 / Formula: EUJ
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-EUJ:
(2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl dioctanoate

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 68.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6z3z

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78370
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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