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- PDB-6oxl: CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECA... -

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Entry
Database: PDB / ID: 6oxl
TitleCRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECAP IN THE PRESENCE OF SS DNA
Components
  • (AP-2 complex subunit ...) x 4
  • Adaptin ear-binding coat-associated protein 2
  • Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain
KeywordsENDOCYTOSIS / AP2 / NECAP2 PROTEIN / CYTOPLASMIC VESICLE / LIPID-BINDING / ADAPTOR / MEMBRANE / TRANSPORT / PHOSPHORYLATION
Function / homology
Function and homology information


clathrin vesicle coat / regulation of vesicle size / clathrin adaptor complex / cardiac septum development / cargo adaptor activity / AP-2 adaptor complex / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / positive regulation of synaptic vesicle endocytosis / membrane coat ...clathrin vesicle coat / regulation of vesicle size / clathrin adaptor complex / cardiac septum development / cargo adaptor activity / AP-2 adaptor complex / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / positive regulation of synaptic vesicle endocytosis / membrane coat / clathrin adaptor activity / coronary vasculature development / vesicle budding from membrane / clathrin coat assembly / signal sequence binding / clathrin-dependent endocytosis / aorta development / postsynaptic neurotransmitter receptor internalization / negative regulation of protein localization to plasma membrane / clathrin binding / ventricular septum development / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / low-density lipoprotein particle receptor binding / regulation of hematopoietic stem cell differentiation / phosphatidylinositol binding / clathrin-coated pit / vesicle-mediated transport / presynapse / receptor internalization / negative regulation of neuron death / secretory granule / intracellular protein transport / protein transport / postsynapse / receptor-mediated endocytosis / endocytosis / synaptic vesicle / protein-containing complex assembly / disordered domain specific binding / heart development / ion channel binding / cytoplasmic vesicle / lipid binding / glutamatergic synapse / protein domain specific binding / intracellular membrane-bounded organelle / protein-containing complex binding / protein kinase binding / mitochondrion / plasma membrane
Clathrin adaptor, mu subunit / Beta-adaptin appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Adaptor protein complex, sigma subunit / AP-1/2/4 complex subunit beta / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Armadillo-type fold / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Coatomer/calthrin adaptor appendage, C-terminal subdomain ...Clathrin adaptor, mu subunit / Beta-adaptin appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Adaptor protein complex, sigma subunit / AP-1/2/4 complex subunit beta / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Armadillo-type fold / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / NECAP, PHear domain / TBP domain superfamily / PH-like domain superfamily / Armadillo-like helical / Clathrin adaptor, mu subunit, conserved site / Longin-like domain superfamily / Adaptin N terminal region / Mu2, C-terminal domain / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / Armadillo / AP-2 complex subunit mu, N-terminal / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / AP-2 complex subunit sigma / Clathrin adaptor complex, small chain / AP complex subunit beta / AP complex, mu/sigma subunit / Protein of unknown function (DUF1681) / Beta-Lactamase - #60 / Mu homology domain, subdomain B / Leucine-rich Repeat Variant / Leucine-rich Repeat Variant / Beta-Lactamase / Alpha Horseshoe / Immunoglobulin-like / Sandwich / 2-Layer Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
AP-2 complex subunit alpha-2 / AP-2 complex subunit beta / Adaptin ear-binding coat-associated protein 2 / AP-2 complex subunit mu / AP-2 complex subunit sigma
Biological speciesMus musculus (house mouse)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPartlow, E.A. / Baker, R.W. / Beacham, G.M. / Chappie, J. / Leschziner, A.E. / Hollopeter, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM127548-01A1 United States
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 13, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: AP-2 complex subunit alpha-2
B: AP-2 complex subunit beta
M: AP-2 complex subunit mu
S: AP-2 complex subunit sigma
N: Adaptin ear-binding coat-associated protein 2
n: Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain


Theoretical massNumber of molelcules
Total (without water)232,6996
Polymers232,6996
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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AP-2 complex subunit ... , 4 types, 4 molecules ABMS

#1: Protein AP-2 complex subunit alpha-2


Mass: 69656.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2a2, Adtab / Plasmid: PACYC_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P17427*PLUS
#2: Protein AP-2 complex subunit beta / AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta ...AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta / Beta-2-adaptin / Beta-adaptin / Clathrin assembly protein complex 2 beta large chain / Plasma membrane adaptor HA2/AP2 adaptin beta subunit


Mass: 66953.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2b1, Clapb1 / Plasmid: PET_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9DBG3
#3: Protein AP-2 complex subunit mu / AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit ...AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit mu / Clathrin assembly protein complex 2 mu medium chain / Clathrin coat assembly protein AP50 / Clathrin coat-associated protein AP50 / Mu2-adaptin / Plasma membrane adaptor AP-2 50 kDa protein


Mass: 49806.688 Da / Num. of mol.: 1 / Mutation: E302K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2m1, Clapm1 / Plasmid: PET_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P84091
#4: Protein AP-2 complex subunit sigma / Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / ...Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / Clathrin assembly protein 2 sigma small chain / Clathrin coat assembly protein AP17 / Clathrin coat-associated protein AP17 / Plasma membrane adaptor AP-2 17 kDa protein / Sigma-adaptin 3b / Sigma2-adaptin


Mass: 17038.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2s1, Ap17, Claps2 / Plasmid: PACYC_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P62744

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Protein / Protein/peptide , 2 types, 2 molecules Nn

#5: Protein Adaptin ear-binding coat-associated protein 2 / NECAP endocytosis-associated protein 2 / NECAP-2


Mass: 28629.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Necap2 / Plasmid: PET-21B / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9D1J1
#6: Protein/peptide Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain


Mass: 613.749 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: PET21-B / Production host: Escherichia coli BL21(DE3) (bacteria)

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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: Phosphorylated AP2-NECAP (mu E302K) co-complex in the presence of ss DNA
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml
Details: Contains an E302K mutation in the mu subunit of AP2.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
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 imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2Leginonimage acquisition
4CTFFINDCTF correction
5GctfCTF correction
6RELIONCTF correction
9UCSF Chimeramodel fitting
11RELIONinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
15Rosettamodel refinement
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324922 / Details: Non-Uniform refinement in cryoSPARC v2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: 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 ...Details: 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
Atomic model building
IDPDB-ID3D fitting-ID
12VGL1
21TQZ1

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