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- PDB-7rpk: Cryo-EM structure of murine Dispatched in complex with Sonic hedgehog -

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Basic information

Entry
Database: PDB / ID: 7rpk
TitleCryo-EM structure of murine Dispatched in complex with Sonic hedgehog
Components
  • Protein dispatched homolog 1
  • Sonic hedgehog protein
KeywordsMEMBRANE PROTEIN / RND transporter / Hedgehog binding / Sterol sensing domain / sodium binding
Function / homology
Function and homology information


positive regulation of skeletal muscle cell proliferation / lateral motor column neuron differentiation / forebrain regionalization / cell proliferation in external granule layer / zona limitans intrathalamica formation / respiratory tube development / positive regulation of neurotrophin production / positive regulation of photoreceptor cell differentiation / right lung development / left lung development ...positive regulation of skeletal muscle cell proliferation / lateral motor column neuron differentiation / forebrain regionalization / cell proliferation in external granule layer / zona limitans intrathalamica formation / respiratory tube development / positive regulation of neurotrophin production / positive regulation of photoreceptor cell differentiation / right lung development / left lung development / primary prostatic bud elongation / : / mesenchymal smoothened signaling pathway involved in prostate gland development / fungiform papilla development / tracheoesophageal septum formation / Release of Hh-Np from the secreting cell / digestive tract mesoderm development / fungiform papilla morphogenesis / striated muscle tissue development / ventral spinal cord interneuron specification / formation of anatomical boundary / bud outgrowth involved in lung branching / patched ligand maturation / mesenchymal-epithelial cell signaling / negative regulation of mesenchymal cell apoptotic process / Ligand-receptor interactions / negative regulation of alpha-beta T cell differentiation / animal organ formation / regulation of prostatic bud formation / hindgut morphogenesis / tongue morphogenesis / male genitalia morphogenesis / positive regulation of striated muscle cell differentiation / mesenchymal cell proliferation / alpha-tubulin acetylation / regulation of epithelial cell proliferation involved in prostate gland development / Activation of SMO / trachea morphogenesis / salivary gland cavitation / telencephalon regionalization / positive regulation of epithelial cell proliferation involved in prostate gland development / anatomical structure formation involved in morphogenesis / trachea development / positive regulation of sclerotome development / negative regulation of ureter smooth muscle cell differentiation / positive regulation of ureter smooth muscle cell differentiation / negative regulation of kidney smooth muscle cell differentiation / positive regulation of kidney smooth muscle cell differentiation / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / embryonic foregut morphogenesis / ectoderm development / epithelial-mesenchymal cell signaling / polarity specification of anterior/posterior axis / lung epithelium development / diaphragm development / spinal cord dorsal/ventral patterning / spinal cord motor neuron differentiation / positive regulation of penile erection / metanephric mesenchymal cell proliferation involved in metanephros development / cholesterol-protein transferase activity / neural tube formation / positive regulation of cerebellar granule cell precursor proliferation / neuron fate specification / positive regulation of oligodendrocyte progenitor proliferation / negative thymic T cell selection / vasculogenesis involved in coronary vascular morphogenesis / laminin-1 binding / negative regulation of T cell differentiation in thymus / myotube differentiation / positive regulation of T cell differentiation in thymus / stem cell development / epithelial cell proliferation involved in salivary gland morphogenesis / tongue development / cerebellar granule cell precursor proliferation / determination of left/right asymmetry in lateral mesoderm / negative regulation of cholesterol efflux / lymphoid progenitor cell differentiation / mesenchymal cell proliferation involved in lung development / Hedgehog ligand biogenesis / intermediate filament organization / lung-associated mesenchyme development / embryonic skeletal system development / branching involved in salivary gland morphogenesis / cell development / limb bud formation / fungiform papilla formation / positive regulation of skeletal muscle tissue development / embryonic hindlimb morphogenesis / embryonic digestive tract morphogenesis / prostate gland development / somite development / male genitalia development / lung lobe morphogenesis / embryonic forelimb morphogenesis / branching involved in prostate gland morphogenesis / hindbrain development / artery development / patched binding / neuron fate commitment
Similarity search - Function
: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Membrane transport protein MMPL domain / MMPL family ...: / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Membrane transport protein MMPL domain / MMPL family / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily
Similarity search - Domain/homology
Chem-6OE / Lauryl Maltose Neopentyl Glycol / CHOLESTEROL HEMISUCCINATE / Protein dispatched homolog 1 / Sonic hedgehog protein
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsAsarnow, D. / Wang, Q. / Ding, K. / Cheng, Y. / Beachy, P.A.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM102498 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM140847 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021741 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2021
Title: Dispatched uses Na flux to power release of lipid-modified Hedgehog.
Authors: Qianqian Wang / Daniel E Asarnow / Ke Ding / Randall K Mann / Jason Hatakeyama / Yunxiao Zhang / Yong Ma / Yifan Cheng / Philip A Beachy /
Abstract: The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters and to H-driven transporters of the RND family, enables tissue-patterning activity of the lipid-modified ...The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters and to H-driven transporters of the RND family, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na gradient across the plasma membrane. The transmembrane channel and Na binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na ions. DISP1-NNN and variants that disrupt single Na sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na-site occupancies, which suggests a mechanism by which transmembrane Na flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na flux powers their conformationally driven activities.
History
DepositionAug 3, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 27, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 24, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 13, 2023Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Revision 1.4Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Protein dispatched homolog 1
H: Sonic hedgehog protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)187,27442
Polymers170,8032
Non-polymers16,47040
Water75742
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3460 Å2
ΔGint-53 kcal/mol
Surface area53210 Å2

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Components

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Protein , 2 types, 2 molecules AH

#1: Protein Protein dispatched homolog 1 / Mdispa


Mass: 152071.141 Da / Num. of mol.: 1 / Fragment: UNP residues 172-1521
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Disp1, Disp, Dispa, Icb, Icbins / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q3TDN0
#2: Protein Sonic hedgehog protein / ShhN / Shh N-terminal processed signaling domains / ShhNp / Sonic hedgehog protein 19 kDa product


Mass: 18732.090 Da / Num. of mol.: 1 / Fragment: UNP residues 26-189
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Shh, Hhg1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q62226

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Sugars , 1 types, 5 molecules

#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 8 types, 77 molecules

#3: Chemical...
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C47H88O22 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-6OE / (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate


Mass: 411.428 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H34NO8P
#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 42 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Dispatched protein complexed with Sonic hedgehog 26-189
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.15191493 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293F
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2300 mMsodium chlorideNaCl1
30.012 % (w/v)lauryl maltoside neopentyl glycolC47H88O221
40.0025 % (w/v)glyo-diosgeninC56H92O251
50.0024 % (w/v)cholesteryl hemisuccinateC31H50O41
65 mMcalcium chlorideCaCl21
SpecimenConc.: 1.56 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: Wait time 20 seconds, blot time 4 seconds, blotting force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Automated data collection in SerialEM using 3x3 image shift pattern (one shot per hole), using beam tilt compensation.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 59880 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1687
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4cryoSPARCCTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11ISOLDEmodel refinement
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1406648
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204786 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 69.9 / Protocol: FLEXIBLE FIT / Space: REAL

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