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- EMDB-53384: Cryo-EM structure of the human NHA2-Fab complex bound to phloretin -

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Basic information

Entry
Database: EMDB / ID: EMD-53384
TitleCryo-EM structure of the human NHA2-Fab complex bound to phloretin
Map dataNHA2_phloretin_J103
Sample
  • Complex: phloretin-bound complex of human NHA2 with Fab
    • Protein or peptide: Fab light chain
    • Protein or peptide: Fab heavy chain
    • Protein or peptide: Sodium/hydrogen exchanger 9B2
  • Ligand: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE
  • Ligand: 3-(4-HYDROXYPHENYL)-1-(2,4,6-TRIHYDROXYPHENYL)PROPAN-1-ONE
KeywordsNa+/H+ exchanger / TRANSPORT PROTEIN
Function / homology
Function and homology information


lithium:proton antiporter activity / lithium ion transport / positive regulation of osteoclast development / sperm principal piece / sodium ion homeostasis / sodium:proton antiporter activity / regulation of insulin secretion involved in cellular response to glucose stimulus / flagellated sperm motility / clathrin-dependent endocytosis / inorganic cation transmembrane transport ...lithium:proton antiporter activity / lithium ion transport / positive regulation of osteoclast development / sperm principal piece / sodium ion homeostasis / sodium:proton antiporter activity / regulation of insulin secretion involved in cellular response to glucose stimulus / flagellated sperm motility / clathrin-dependent endocytosis / inorganic cation transmembrane transport / sodium ion transport / mitochondrial membrane / recycling endosome / Stimuli-sensing channels / synaptic vesicle membrane / recycling endosome membrane / monoatomic ion transmembrane transport / basolateral plasma membrane / mitochondrial inner membrane / apical plasma membrane / mitochondrion / identical protein binding / plasma membrane
Similarity search - Function
: / Sodium/solute symporter superfamily / Cation/H+ exchanger / Sodium/hydrogen exchanger, transmembrane
Similarity search - Domain/homology
Sodium/hydrogen exchanger 9B2
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsJung S / Drew D
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)820187European Union
CitationJournal: Int J Mol Sci / Year: 2025
Title: Structure and Inhibition of the Human Na/H Exchanger SLC9B2.
Authors: Sukkyeong Jung / Surabhi Kokane / Hang Li / So Iwata / Norimichi Nomura / David Drew /
Abstract: The sodium/proton exchanger NHA2, also known as SLC9B2, is important for insulin secretion, renal blood pressure regulation, and electrolyte retention. Recent structures of bison NHA2 has revealed ...The sodium/proton exchanger NHA2, also known as SLC9B2, is important for insulin secretion, renal blood pressure regulation, and electrolyte retention. Recent structures of bison NHA2 has revealed its unique 14-transmembrane helix architecture, which is different from SLC9A/NHE members made up from 13-TM helices. Sodium/proton exchangers are functional homodimers, and the additional N-terminal helix in NHA2 was found to alter homodimer assembly. Here, we present the cryo-electron microscopy structures of apo human NHA2 in complex with a Fab fragment and also with the inhibitor phloretin bound at 2.8 and 2.9 Å resolution, respectively. We show how phosphatidic acid (PA) lipids bind to the homodimer interface of NHA2 on the extracellular side, which we propose has a regulatory role linked to cell volume regulation. The ion binding site of human NHA2 has a salt bridge interaction between the ion binding aspartate D278 and R432, an interaction previously broken in the bison NHA2 structure, and these differences suggest a possible ion coupling mechanism. Lastly, the human NHA2 structure in complex with phloretin offers a template for structure-guided drug design, potentially leading to the development of more selective and potent NHA2 inhibitors.
History
DepositionApr 11, 2025-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53384.png

Downloads & links

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Map

FileDownload / File: emd_53384.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNHA2_phloretin_J103
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 400 pix.
= 260. Å		0.65 Å/pix.
x 400 pix.
= 260. Å		0.65 Å/pix.
x 400 pix.
= 260. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.027
Minimum - Maximum-0.04278281 - 0.09492611
Average (Standard dev.)0.0002117367 (±0.0032334134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 260.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A NHA2 phloretin J103

Fileemd_53384_half_map_1.map
Annotationhalf_map_A_NHA2_phloretin_J103
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B NHA2 phloretin J103

Fileemd_53384_half_map_2.map
Annotationhalf_map_B_NHA2_phloretin_J103
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : phloretin-bound complex of human NHA2 with Fab

EntireName: phloretin-bound complex of human NHA2 with Fab
Components
  • Complex: phloretin-bound complex of human NHA2 with Fab
    • Protein or peptide: Fab light chain
    • Protein or peptide: Fab heavy chain
    • Protein or peptide: Sodium/hydrogen exchanger 9B2
  • Ligand: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE
  • Ligand: 3-(4-HYDROXYPHENYL)-1-(2,4,6-TRIHYDROXYPHENYL)PROPAN-1-ONE

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Supramolecule #1: phloretin-bound complex of human NHA2 with Fab

SupramoleculeName: phloretin-bound complex of human NHA2 with Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Fab light chain

MacromoleculeName: Fab light chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.715109 KDa
SequenceString: DIVMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWFQQKP DGTVKLLICY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQ QDSKHPWTFG GGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String:
DIVMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWFQQKP DGTVKLLICY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQ QDSKHPWTFG GGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC

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Macromolecule #2: Fab heavy chain

MacromoleculeName: Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 27.757529 KDa
SequenceString: EVQLQESGPE LVKPGASVKM SCKASGYTFT NYFIHWVKQK PGQGLEWIGY INPYNDITKF NEKFKGKATL TSDKSSRTAY MELSSLTSE DSAVYYCARC DGYYRYYAMD YWGQGTSVTV SSAKTTAPSV YPLAPVCGDT SGSSVTLGCL VKGYFPEPVT L TWNSGSLS ...String:
EVQLQESGPE LVKPGASVKM SCKASGYTFT NYFIHWVKQK PGQGLEWIGY INPYNDITKF NEKFKGKATL TSDKSSRTAY MELSSLTSE DSAVYYCARC DGYYRYYAMD YWGQGTSVTV SSAKTTAPSV YPLAPVCGDT SGSSVTLGCL VKGYFPEPVT L TWNSGSLS SGVHTFPAVL QSDLYTLSSS VTVTSSTWPS QSITCNVAHP ASSTKVDKKI EPRGPTIKPC PPCKCPAPNL LG GPSVFIF PPKIKDVLM

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Macromolecule #3: Sodium/hydrogen exchanger 9B2

MacromoleculeName: Sodium/hydrogen exchanger 9B2 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.611672 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGDEDKRITY EDSEPSTGMN YTPSMHQEAQ EETVMKLKGI DANEPTEGSI LLKSSEKKLQ ETPTEANHVQ RLRQMLACPP HGLLDRVIT NVTIIVLLWA VVWSITGSEC LPGGNLFGII ILFYCAIIGG KLLGLIKLPT LPPLPSLLGM LLAGFLIRNI P VINDNVQI ...String:
MGDEDKRITY EDSEPSTGMN YTPSMHQEAQ EETVMKLKGI DANEPTEGSI LLKSSEKKLQ ETPTEANHVQ RLRQMLACPP HGLLDRVIT NVTIIVLLWA VVWSITGSEC LPGGNLFGII ILFYCAIIGG KLLGLIKLPT LPPLPSLLGM LLAGFLIRNI P VINDNVQI KHKWSSSLRS IALSIILVRA GLGLDSKALK KLKGVCVRLS MGPCIVEACT SALLAHYLLG LPWQWGFILG FV LGAVSPA VVVPSMLLLQ GGGYGVEKGV PTLLMAAGSF DDILAITGFN TCLGIAFSTG STVFNVLRGV LEVVIGVATG SVL GFFIQY FPSRDQDKLV CKRTFLVLGL SVLAVFSSVH FGFPGSGGLC TLVMAFLAGM GWTSEKAEVE KIIAVAWDIF QPLL FGLIG AEVSIASLRP ETVGLCVATV GIAVLIRILT TFLMVCFAGF NLKEKIFISF AWLPKATVQA AIGSVALDTA RSHGE KQLE DYGMDVLTVA FLSILITAPI GSLLIGLLGP RLLQKVEHQN KDEEVQGETS VQV

UniProtKB: Sodium/hydrogen exchanger 9B2

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Macromolecule #4: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE

MacromoleculeName: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE
type: ligand / ID: 4 / Number of copies: 2 / Formula: LPP
Molecular weightTheoretical: 648.891 Da
Chemical component information

ChemComp-LPP:
2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE / phospholipid*YM

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Macromolecule #5: 3-(4-HYDROXYPHENYL)-1-(2,4,6-TRIHYDROXYPHENYL)PROPAN-1-ONE

MacromoleculeName: 3-(4-HYDROXYPHENYL)-1-(2,4,6-TRIHYDROXYPHENYL)PROPAN-1-ONE
type: ligand / ID: 5 / Number of copies: 2 / Formula: G50
Molecular weightTheoretical: 274.269 Da
Chemical component information

ChemComp-G50:
3-(4-HYDROXYPHENYL)-1-(2,4,6-TRIHYDROXYPHENYL)PROPAN-1-ONE

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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: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 65.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 251355
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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