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- PDB-7b2l: Structure of the endocytic adaptor complex AENTH -

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Basic information

Entry
Database: PDB / ID: 7b2l
TitleStructure of the endocytic adaptor complex AENTH
Components
  • ANTH domain of Sla2
  • ENTH domain of epsin Ent1
KeywordsCELL ADHESION / Membrane protein / clathrin-mediated endocytosis / adapter protein / Sla2 / Epsin-1 / ENTH / ANTH
Function / homology
Function and homology information


Cargo recognition for clathrin-mediated endocytosis / actin cortical patch assembly / clathrin vesicle coat / clathrin light chain binding / negative regulation of Arp2/3 complex-mediated actin nucleation / actin cortical patch / incipient cellular bud site / cellular bud tip / clathrin coat assembly / clathrin adaptor activity ...Cargo recognition for clathrin-mediated endocytosis / actin cortical patch assembly / clathrin vesicle coat / clathrin light chain binding / negative regulation of Arp2/3 complex-mediated actin nucleation / actin cortical patch / incipient cellular bud site / cellular bud tip / clathrin coat assembly / clathrin adaptor activity / cellular bud neck / mating projection tip / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / clathrin-coated vesicle / clathrin binding / ubiquitin binding / actin filament organization / phospholipid binding / endocytosis / actin filament binding / early endosome / endosome / plasma membrane / cytoplasm
Similarity search - Function
Sla2 family / AP180 N-terminal homology (ANTH) domain / ANTH domain / ENTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain ...Sla2 family / AP180 N-terminal homology (ANTH) domain / ANTH domain / ENTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain / I/LWEQ domain profile. / I/LWEQ domain / ENTH/VHS / Ubiquitin-interacting motif. / Ubiquitin interacting motif / Ubiquitin-interacting motif (UIM) domain profile.
Similarity search - Domain/homology
Chem-PIO / Protein SLA2 / Epsin-1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKlebl, D.P. / Lizarrondo, J. / Sobott, F. / Garcia-Alai, M. / Muench, S.P.
Funding support Germany, United Kingdom, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)SK 305/1-1 Germany
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis.
Authors: Javier Lizarrondo / David P Klebl / Stephan Niebling / Marc Abella / Martin A Schroer / Haydyn D T Mertens / Katharina Veith / Roland Thuenauer / Dmitri I Svergun / Michal Skruzny / Frank ...Authors: Javier Lizarrondo / David P Klebl / Stephan Niebling / Marc Abella / Martin A Schroer / Haydyn D T Mertens / Katharina Veith / Roland Thuenauer / Dmitri I Svergun / Michal Skruzny / Frank Sobott / Stephen P Muench / Maria M Garcia-Alai /
Abstract: During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat ...During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.
History
DepositionNov 27, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: ENTH domain of epsin Ent1
B: ANTH domain of Sla2
C: ENTH domain of epsin Ent1
D: ANTH domain of Sla2
F: ENTH domain of epsin Ent1
G: ANTH domain of Sla2
H: ENTH domain of epsin Ent1
I: ANTH domain of Sla2
K: ENTH domain of epsin Ent1
L: ANTH domain of Sla2
M: ENTH domain of epsin Ent1
N: ANTH domain of Sla2
P: ENTH domain of epsin Ent1
Q: ANTH domain of Sla2
R: ENTH domain of epsin Ent1
S: ANTH domain of Sla2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)432,03136
Polymers417,10016
Non-polymers14,93120
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ENTH domain of epsin Ent1


Mass: 18880.510 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: ENTH domain of epsin Ent1 / Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ENT1, YDL161W / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q12518
#2: Protein
ANTH domain of Sla2 / ANTH domain of Sla2


Mass: 33257.004 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: ANTH domain of Sla2 / Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SLA2, END4, MOP2, UFG1, YNL243W, N1102 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P33338
#3: Chemical
ChemComp-PIO / [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: C25H49O19P3 / Feature type: SUBJECT OF INVESTIGATION
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: 16 mer of 8 Sla2 ANTH and 8 Epsin-1 ENTH domains in complex with PIP2
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 20 mM Tris, 250 mM NaCl and 1 mM DTT
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 278 K

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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: 75.2 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9ISOLDEmodel refinement
10PHENIXmodel refinement
11RELIONinitial Euler assignment
12cryoSPARCfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 195536
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79414 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-ID
15OO7

5oo7

1
25ONF

5onf

1

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