MEMBRANE PROTEIN / G-protein coupled receptor / palmitoleation / secretion / embryonic development
Function / homology
Function and homology information
: / : / Wnt protein secretion / neural crest cell fate commitment / positive regulation of Wnt protein secretion / WNT ligand biogenesis and trafficking / secondary palate development / cementum mineralization / beta-catenin destruction complex disassembly / hindbrain development ...: / : / Wnt protein secretion / neural crest cell fate commitment / positive regulation of Wnt protein secretion / WNT ligand biogenesis and trafficking / secondary palate development / cementum mineralization / beta-catenin destruction complex disassembly / hindbrain development / Wnt-protein binding / exocrine pancreas development / frizzled binding / Class B/2 (Secretin family receptors) / Disassembly of the destruction complex and recruitment of AXIN to the membrane / anterior/posterior axis specification / midbrain development / mesoderm formation / positive regulation of Wnt signaling pathway / canonical Wnt signaling pathway / cell fate commitment / endomembrane system / response to retinoic acid / TCF dependent signaling in response to WNT / cytokine activity / intracellular protein transport / neuron differentiation / trans-Golgi network / Wnt signaling pathway / endocytic vesicle membrane / positive regulation of canonical Wnt signaling pathway / early endosome membrane / cytoplasmic vesicle / positive regulation of canonical NF-kappaB signal transduction / collagen-containing extracellular matrix / receptor ligand activity / early endosome / Golgi membrane / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular exosome / extracellular region / identical protein binding / plasma membrane / cytosol Similarity search - Function
Wnt-8 protein / Protein Wnt-8A/8C / Protein wntless / : / Wntless-like, transmembrane domain / Wnt protein, conserved site / Wnt-1 family signature. / Wnt / Wnt, C-terminal domain / wnt family / found in Wnt-1 Similarity search - Domain/homology
Chem-DR9 / PALMITIC ACID / CHOLESTEROL HEMISUCCINATE / Protein wntless homolog / Protein Wnt-8a Similarity search - Component
Biological species
Homo sapiens (human)
Method
ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35 GM132120
United States
National Research Foundation (NRF, Singapore)
MOH-000155
Singapore
Citation
Journal: Cell / Year: 2021 Title: Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion. Authors: Rie Nygaard / Jia Yu / Jonathan Kim / Daniel R Ross / Giacomo Parisi / Oliver B Clarke / David M Virshup / Filippo Mancia / Abstract: Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O- ...Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
EMPIAR-10699 (Title: Human WLS in complex with WNT8A / Data size: 1.9 TB Data #1: Unaligned multi frame micrographs of WNT8A WLS complex [micrographs - multiframe])
Mass: 40052.359 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: WNT8A, WNT8D / Plasmid: pEG BacMam vector / Cell line (production host): FreeStyle HEK293-F Cells / Production host: Homo sapiens (human) / References: UniProt: Q9H1J5
#2: Protein
Proteinwntlesshomolog / Integral membrane protein GPR177 / Protein evenness interrupted homolog / EVI / Putative NF-kappa-B- ...Integral membrane protein GPR177 / Protein evenness interrupted homolog / EVI / Putative NF-kappa-B-activating protein 373
Mass: 65649.430 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: WLS, C1orf139, GPR177, UNQ85/PRO18667 / Plasmid: pEG BacMam vector / Cell line (production host): FreeStyle HEK293-F cells / Production host: Homo sapiens (human) / References: UniProt: Q5T9L3
Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
-
Details
Has ligand of interest
Y
-
Experimental details
-
Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
-
Sample preparation
Component
Name: Complex of WLS/Evi and WNT8A in nanodisc / Type: COMPLEX / Details: Nanodisc were formed using MSP1E3D1 and POPG lipid / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weight
Value: 0.1056 MDa / Experimental value: NO
Source (natural)
Organism: Homo sapiens (human)
Source (recombinant)
Organism: Homo sapiens (human) / Strain: FreeStyle 293-F Cells
Buffer solution
pH: 8
Buffer component
ID
Conc.
Name
Formula
Buffer-ID
1
100mM
Tris
1
2
150mM
NaCl
1
Specimen
Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
-
Electron microscopy imaging
Microscopy
Model: FEI TITAN
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD
Image recording
Average exposure time: 3 sec. / Electron dose: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12747
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