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- EMDB-33865: The Cryo-EM Structure of Human Tissue Nonspecific Alkaline Phosph... -

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Basic information

Entry
Database: EMDB / ID: EMD-33865
TitleThe Cryo-EM Structure of Human Tissue Nonspecific Alkaline Phosphatase and Single-Chain Fragment Variable (ScFv) Complex.
Map data
Sample
  • Complex: hALPL-scFv
    • Protein or peptide: Alkaline phosphatase, tissue-nonspecific isozyme
    • Protein or peptide: scFvSingle-chain variable fragment
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium
Keywordsalkaline phosphatase / bone mineralization / catalytic network / hypophosphatasia / structural biology / HYDROLASE
Function / homology
Function and homology information


phosphoamidase / pyridoxal phosphate metabolic process / phosphoamidase activity / phosphoethanolamine phosphatase activity / response to vitamin B6 / futile creatine cycle / pyridoxal phosphatase activity / developmental process involved in reproduction / ADP phosphatase activity / response to macrophage colony-stimulating factor ...phosphoamidase / pyridoxal phosphate metabolic process / phosphoamidase activity / phosphoethanolamine phosphatase activity / response to vitamin B6 / futile creatine cycle / pyridoxal phosphatase activity / developmental process involved in reproduction / ADP phosphatase activity / response to macrophage colony-stimulating factor / inhibition of non-skeletal tissue mineralization / pyrophosphatase activity / Post-translational modification: synthesis of GPI-anchored proteins / cementum mineralization / alkaline phosphatase / alkaline phosphatase activity / response to sodium phosphate / phosphate ion homeostasis / inorganic diphosphate phosphatase activity / cellular homeostasis / endochondral ossification / response to vitamin D / bone mineralization / cellular response to organic cyclic compound / calcium ion homeostasis / side of membrane / dephosphorylation / response to glucocorticoid / extracellular matrix / skeletal system development / mitochondrial membrane / response to insulin / mitochondrial intermembrane space / osteoblast differentiation / positive regulation of cold-induced thermogenesis / response to lipopolysaccharide / response to antibiotic / calcium ion binding / ATP hydrolysis activity / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
Alkaline phosphatase, active site / Alkaline phosphatase active site. / Alkaline phosphatase / Alkaline phosphatase / Alkaline phosphatase homologues / Alkaline-phosphatase-like, core domain superfamily
Similarity search - Domain/homology
Alkaline phosphatase, tissue-nonspecific isozyme
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsYu YT / Yao DQ / Zhang Q / Rao B / Xia Y / Lu Y / Qin A / Ma PX / Cao Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2023
Title: The structural pathology for hypophosphatasia caused by malfunctional tissue non-specific alkaline phosphatase.
Authors: Yating Yu / Kewei Rong / Deqiang Yao / Qing Zhang / Xiankun Cao / Bing Rao / Ying Xia / Yi Lu / Yafeng Shen / Ying Yao / Hongtao Xu / Peixiang Ma / Yu Cao / An Qin /
Abstract: Hypophosphatasia (HPP) is a metabolic bone disease that manifests as developmental abnormalities in bone and dental tissues. HPP patients exhibit hypo-mineralization and osteopenia due to the ...Hypophosphatasia (HPP) is a metabolic bone disease that manifests as developmental abnormalities in bone and dental tissues. HPP patients exhibit hypo-mineralization and osteopenia due to the deficiency or malfunction of tissue non-specific alkaline phosphatase (TNAP), which catalyzes the hydrolysis of phosphate-containing molecules outside the cells, promoting the deposition of hydroxyapatite in the extracellular matrix. Despite the identification of hundreds of pathogenic TNAP mutations, the detailed molecular pathology of HPP remains unclear. Here, to address this issue, we determine the crystal structures of human TNAP at near-atomic resolution and map the major pathogenic mutations onto the structure. Our study reveals an unexpected octameric architecture for TNAP, which is generated by the tetramerization of dimeric TNAPs, potentially stabilizing the TNAPs in the extracellular environments. Moreover, we use cryo-electron microscopy to demonstrate that the TNAP agonist antibody (JTALP001) forms a stable complex with TNAP by binding to the octameric interface. The administration of JTALP001 enhances osteoblast mineralization and promoted recombinant TNAP-rescued mineralization in TNAP knockout osteoblasts. Our findings elucidate the structural pathology of HPP and highlight the therapeutic potential of the TNAP agonist antibody for osteoblast-associated bone disorders.
History
DepositionJul 18, 2022-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33865.png

Downloads & links

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Map

FileDownload / File: emd_33865.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-3.8920383 - 6.6240916
Average (Standard dev.)0.0013514833 (±0.14232546)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33865_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33865_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : hALPL-scFv

EntireName: hALPL-scFv
Components
  • Complex: hALPL-scFv
    • Protein or peptide: Alkaline phosphatase, tissue-nonspecific isozyme
    • Protein or peptide: scFvSingle-chain variable fragment
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: hALPL-scFv

SupramoleculeName: hALPL-scFv / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Alkaline phosphatase, tissue-nonspecific isozyme

MacromoleculeName: Alkaline phosphatase, tissue-nonspecific isozyme / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: alkaline phosphatase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.020188 KDa
Recombinant expressionOrganism: Trichopalpus nigribasis (fry)
SequenceString: MKTIIALSYI FCLVFAGRAL VPEKEKDPKY WRDQAQETLK YALELQKLNT NVAKNVIMFL GDGMGVSTVT AARILKGQLH HNPGEETRL EMDKFPFVAL SKTYNTNAQV PDSAGTATAY LCGVKANEGT VGVSAATERS RCNTTQGNEV TSILRWAKDA G KSVGIVTT ...String:
MKTIIALSYI FCLVFAGRAL VPEKEKDPKY WRDQAQETLK YALELQKLNT NVAKNVIMFL GDGMGVSTVT AARILKGQLH HNPGEETRL EMDKFPFVAL SKTYNTNAQV PDSAGTATAY LCGVKANEGT VGVSAATERS RCNTTQGNEV TSILRWAKDA G KSVGIVTT TRVNHATPSA AYAHSADRDW YSDNEMPPEA LSQGCKDIAY QLMHNIRDID VIMGGGRKYM YPKNKTDVEY ES DEKARGT RLDGLDLVDT WKSFKPRYKH SHFIWNRTEL LTLDPHNVDY LLGLFEPGDM QYELNRNNVT DPSLSEMVVV AIQ ILRKNP KGFFLLVEGG RIDHGHHEGK AKQALHEAVE MDRAIGQAGS LTSSEDTLTV VTADHSHVFT FGGYTPRGNS IFGL APMLS DTDKKPFTAI LYGNGPGYKV VGGERENVSM VDYAHNNYQA QSAVPLRHET HGGEDVAVFS KGPMAHLLHG VHEQN YVPH VMAYAACIGA NLGHCAPAAA AENLYFQGCC PGCC

UniProtKB: Alkaline phosphatase, tissue-nonspecific isozyme

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Macromolecule #2: scFv

MacromoleculeName: scFv / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.830195 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: MYRMQLLSCI ALSLALVTNS AAQPAMAQVQ LVQSGAEVKK PGASVKVSCK ASGYTFTSYG ISWVRQAPGQ GLEWMGWISA YNGNTNYAQ KLQGRVTMTT DTSTSTAYME LRSLRSDDTA VYYCARDKGW NSEGSLEYWG QGTLVTVSSG GGGSGGGGSG G GGSDIVMT ...String:
MYRMQLLSCI ALSLALVTNS AAQPAMAQVQ LVQSGAEVKK PGASVKVSCK ASGYTFTSYG ISWVRQAPGQ GLEWMGWISA YNGNTNYAQ KLQGRVTMTT DTSTSTAYME LRSLRSDDTA VYYCARDKGW NSEGSLEYWG QGTLVTVSSG GGGSGGGGSG G GGSDIVMT QSPSSVSASV GDRVTITCRA SQGISNWLGW YQQKPGKAPK LLIYGASSLQ SGVPSRFSGS GSGTDFTLTI SS LQPEDFA TYFCQQAYSL PLTFGGGTKL EIKRGAAA

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 314858
FSC plot (resolution estimation)

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