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- PDB-7e2h: Cryo-EM structure of hDisp1NNN-3C-Cleavage -

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Basic information

Entry
Database: PDB / ID: 7e2h
TitleCryo-EM structure of hDisp1NNN-3C-Cleavage
Components(Protein dispatched homolog 1) x 2
KeywordsLIPID TRANSPORT / membrane protein
Function / homology
Function and homology information


patched ligand maturation / diaphragm development / embryonic pattern specification / peptide transport / peptide transmembrane transporter activity / dorsal/ventral pattern formation / determination of left/right symmetry / smoothened signaling pathway / regulation of protein secretion / protein homotrimerization ...patched ligand maturation / diaphragm development / embryonic pattern specification / peptide transport / peptide transmembrane transporter activity / dorsal/ventral pattern formation / determination of left/right symmetry / smoothened signaling pathway / regulation of protein secretion / protein homotrimerization / basolateral plasma membrane / membrane
Similarity search - Function
Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
CHOLESTEROL HEMISUCCINATE / Protein dispatched homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 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
D: Protein dispatched homolog 1
E: Protein dispatched homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)174,70414
Polymers169,9252
Non-polymers4,77912
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7290 Å2
ΔGint-48 kcal/mol
Surface area44550 Å2
MethodPISA

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Components

#1: Protein Protein dispatched homolog 1


Mass: 29497.619 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DISP1, DISPA
Production host: Mammalian expression vector EGFP-MCS-pcDNA3.1 (others)
References: UniProt: Q96F81
#2: Protein Protein dispatched homolog 1


Mass: 140427.594 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 EGFP-MCS-pcDNA3.1 (others)
References: UniProt: Q96F81
#3: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C31H50O4
#4: 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 / N-Acetylglucosamine


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

ComponentName: hDisp1NNN-3C / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalian expression vector EGFP-MCS-pcDNA3.1 (others)
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 FIELDBright-field microscopy
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: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63043 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037483
ELECTRON MICROSCOPYf_angle_d0.65510247
ELECTRON MICROSCOPYf_dihedral_angle_d8.14414
ELECTRON MICROSCOPYf_chiral_restr0.0631215
ELECTRON MICROSCOPYf_plane_restr0.0041241

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