National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM127548-01A1
United States
Citation
Journal: Elife / Year: 2019 Title: A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex. Authors: Edward A Partlow / Richard W Baker / Gwendolyn M Beacham / Joshua S Chappie / Andres E Leschziner / Gunther Hollopeter / Abstract: Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma ...Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in . Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.
History
Deposition
May 10, 2019
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Header (metadata) release
Aug 28, 2019
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Map release
Sep 11, 2019
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Update
Dec 2, 2020
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Current status
Dec 2, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: 4 UL OF SAMPLE/GRID WAS BLOTTED FOR 4 SECONDS AND PLUNGE FROZEN IN LIQUID-NITROGEN COOLED ETHANE.
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Software
Name: Leginon
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 944 / Average electron dose: 1.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
STARTING MODEL WAS GENERATED BY DOCKING PDB ENTRIES 2VGL AND 1TQZ INTO CRYO-EM DENSITY AND MANUALLY EDITING SEQUENCE AND STRUCTURAl CHANGES. MODEL REFINEMENT WAS PERFORMED USING ROSETTA AND PHENIX.REAL_SPACE_REFINE
Refinement
Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 63 / Target criteria: MAXIMUM LIKELIHOOD
Output model
PDB-6owo: CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 CORE BOUND TO NECAP
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