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- PDB-7e2i: Cryo-EM structure of hDisp1NNN-ShhN -

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Basic information

Entry
Database: PDB / ID: 7e2i
TitleCryo-EM structure of hDisp1NNN-ShhN
Components
  • Protein dispatched homolog 1
  • Sonic hedgehog protein
KeywordsLIPID TRANSPORT / membrane protein
Function / homology
Function and homology information


patched ligand maturation / morphogen activity / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation ...patched ligand maturation / morphogen activity / positive regulation of skeletal muscle cell proliferation / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / 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 / hindgut morphogenesis / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / trunk neural crest cell migration / Formation of lateral plate mesoderm / polarity specification of anterior/posterior axis / striated muscle tissue development / negative regulation of alpha-beta T cell differentiation / regulation of glial cell proliferation / regulation of prostatic bud formation / ventral midline development / metanephric mesenchymal cell proliferation involved in metanephros development / formation of anatomical boundary / lung epithelium development / cholesterol-protein transferase activity / positive regulation of striated muscle cell differentiation / trachea morphogenesis / bud outgrowth involved in lung branching / HHAT G278V doesn't palmitoylate Hh-Np / diaphragm development / telencephalon regionalization / epithelial-mesenchymal cell signaling / Ligand-receptor interactions / laminin-1 binding / limb bud formation / lung lobe morphogenesis / negative regulation of cholesterol efflux / salivary gland cavitation / spinal cord dorsal/ventral patterning / negative regulation of mesenchymal cell apoptotic process / embryonic digestive tract morphogenesis / determination of left/right asymmetry in lateral mesoderm / epithelial cell proliferation involved in salivary gland morphogenesis / positive regulation of cerebellar granule cell precursor proliferation / negative regulation of T cell differentiation in thymus / spinal cord motor neuron differentiation / cell development / positive regulation of T cell differentiation in thymus / Activation of SMO / mesenchymal cell proliferation involved in lung development / cerebellar granule cell precursor proliferation / intermediate filament organization / prostate gland development / lymphoid progenitor cell differentiation / embryonic skeletal system development / establishment of epithelial cell polarity / skeletal muscle fiber differentiation / somite development / mesenchymal cell apoptotic process / patched binding / neuron fate commitment / animal organ formation / embryonic foregut morphogenesis / hindbrain development / ectoderm development / positive regulation of skeletal muscle tissue development / stem cell development / embryonic pattern specification / branching involved in salivary gland morphogenesis / thalamus development / negative regulation of dopaminergic neuron differentiation / skeletal muscle cell proliferation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / positive regulation of immature T cell proliferation in thymus / dorsal/ventral neural tube patterning / negative thymic T cell selection / smooth muscle tissue development / self proteolysis / pattern specification process / male genitalia development / artery development / lung-associated mesenchyme development / positive regulation of astrocyte differentiation / oligodendrocyte development / regulation of stem cell proliferation / epithelial cell proliferation involved in prostate gland development / positive regulation of epithelial cell proliferation involved in prostate gland development / Release of Hh-Np from the secreting cell / dopaminergic neuron differentiation / intein-mediated protein splicing / glycosaminoglycan binding / regulation of proteolysis / Formation of axial mesoderm / metanephric collecting duct development / metanephros development / positive thymic T cell selection
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 / Protein patched/dispatched / Patched 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 / Protein patched/dispatched / Patched family / 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
CHOLESTEROL HEMISUCCINATE / Sonic hedgehog protein / Protein dispatched homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.07 Å
AuthorsLi, W. / Wang, L. / Gong, X.
Funding support China, 2items
OrganizationGrant numberCountry
Other government China
Other government China
CitationJournal: Nat Commun / Year: 2021
Title: Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release.
Authors: Wanqiu Li / Linlin Wang / Bradley M Wierbowski / Mo Lu / Feitong Dong / Wenchen Liu / Sisi Li / Peiyi Wang / Adrian Salic / Xin Gong /
Abstract: The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- ...The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand. These structures, together with biochemical data, reveal that proteolytic cleavage opens the extracellular domain of hDisp1, removing steric hindrance to Shh binding. Structure-guided functional experiments demonstrate the role of hDisp1-Shh interactions in ligand release. Our results clarify the mechanisms of hDisp1 activation and Shh morphogen release, and highlight how a unique proteolytic cleavage event enabled acquisition of a protein substrate by a member of a family of small molecule transporters.
History
DepositionFeb 5, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
G: Sonic hedgehog protein
D: Protein dispatched homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,14414
Polymers220,7862
Non-polymers4,35712
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2340 Å2
ΔGint0 kcal/mol
Surface area51940 Å2

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Components

#1: Protein Sonic hedgehog protein / SHH / HHG-1 / Shh unprocessed N-terminal signaling and C-terminal autoprocessing domains / ShhNC


Mass: 49676.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHH
Production host: Mammalian expression vector Flag-EGFP-MCS-pcDNA3.1 (others)
References: UniProt: Q15465
#2: Protein Protein dispatched homolog 1


Mass: 171110.094 Da / Num. of mol.: 1 / Mutation: D572N,D573N,D1051N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DISP1, DISPA
Production host: Mammalian expression vector Flag-EGFP-MCS-pcDNA3.1 (others)
References: UniProt: Q96F81
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C31H50O4
#5: 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.: 4 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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
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

Component
IDNameTypeEntity IDParent-IDSource
1hDisp1NNN-ShhNCOMPLEX#1-#20RECOMBINANT
2ShhNCOMPLEX#11RECOMBINANT
3hDisp1NNNCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mammalian expression vector EGFP-MCS-pcDNA3.1 (others)2021194
33Mammalian expression vector EGFP-MCS-pcDNA3.1 (others)2021194
Buffer solutionpH: 8
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173169 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0068806
ELECTRON MICROSCOPYf_angle_d0.76912015
ELECTRON MICROSCOPYf_dihedral_angle_d9.1295212
ELECTRON MICROSCOPYf_chiral_restr0.0541387
ELECTRON MICROSCOPYf_plane_restr0.0051476

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