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- PDB-6d83: Structure of the cargo bound AP-1:Arf1:tetherin-Nef (L164A, L165A... -

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Entry
Database: PDB / ID: 6d83
TitleStructure of the cargo bound AP-1:Arf1:tetherin-Nef (L164A, L165A) dileucine mutant dimer monomeric subunit
Components
  • (AP-1 complex subunit ...AP-1 transcription factor) x 4
  • ADP-ribosylation factor 1ARF1
  • Bone marrow stromal antigen 2,Protein Nef
KeywordsPROTEIN TRANSPORT / AP / HIV / Nef / trafficking
Function / homologyAdaptin C-terminal domain / HIV negative factor Nef / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / TBP domain superfamily / Synthesis of PIPs at the plasma membrane / Longin-like domain superfamily / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Gamma-adaptin ear (GAE) domain / Lysosome Vesicle Biogenesis / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain ...Adaptin C-terminal domain / HIV negative factor Nef / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / TBP domain superfamily / Synthesis of PIPs at the plasma membrane / Longin-like domain superfamily / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Gamma-adaptin ear (GAE) domain / Lysosome Vesicle Biogenesis / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Small GTPase superfamily, ARF/SAR type / Clathrin derived vesicle budding / Small GTP-binding protein domain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, mu subunit / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor complex, small chain / Armadillo / MHC class II antigen presentation / Nef Mediated CD4 Down-regulation / Synthesis of PIPs at the Golgi membrane / Bone marrow stromal antigen 2 / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor complexes medium chain signature 2. / Gamma-adaptin ear (GAE) domain profile. / Mu homology domain (MHD) profile. / small GTPase Arf family profile. / Nef mediated downregulation of MHC class I complex cell surface expression / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Armadillo-like helical / Armadillo-type fold / Mu homology domain / Neutrophil degranulation / Adaptin N terminal region / Golgi Associated Vesicle Biogenesis / Clathrin adaptor complex small chain / Lysosome Vesicle Biogenesis / Adaptor complexes medium subunit family / Negative factor, (F-Protein) or Nef / MHC class II antigen presentation / Interferon alpha/beta signaling / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / AP-1/2/4 complex subunit beta / ADP-ribosylation factor family / AP-2 complex subunit mu, C-terminal superfamily / Beta2-adaptin appendage, C-terminal sub-domain / HIV-1 Nef protein, core domain superfamily / AP complex subunit beta / HIV-1 Nef protein, anchor domain superfamily / Adaptor protein complex, sigma subunit / COPI-dependent Golgi-to-ER retrograde traffic / Adaptor protein complex AP-1, gamma subunit / COPI-mediated anterograde transport / Neutrophil degranulation / P-loop containing nucleoside triphosphate hydrolase / Clathrin adaptor, mu subunit, conserved site / Golgi Associated Vesicle Biogenesis / AP complex, mu/sigma subunit / Intra-Golgi traffic / Bone marrow stromal antigen 2 / clathrin adaptor complex / Golgi to transport vesicle transport / synaptic vesicle budding / lysosomal membrane organization / negative regulation of intracellular transport of viral material / negative regulation of plasmacytoid dendritic cell cytokine production / positive regulation of late endosome to lysosome transport / phospholipase D activator activity / regulation of phospholipid metabolic process / positive regulation of natural killer cell degranulation / negative regulation by symbiont of host T-cell mediated immune response / negative regulation of CD4 biosynthetic process / regulation of Arp2/3 complex-mediated actin nucleation / mitotic cleavage furrow ingression / AP-1 adaptor complex / endosome to melanosome transport / positive regulation of ER to Golgi vesicle-mediated transport / Golgi to lysosome transport / response to interferon-beta / very-low-density lipoprotein particle assembly / melanosome organization / regulation of receptor internalization / postsynaptic actin cytoskeleton organization / suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I / COPI-coated vesicle / response to interferon-alpha / dendritic spine organization / GTP-dependent protein binding / membrane coat / suppression by virus of host antigen processing and presentation of peptide antigen via MHC class II / suppression by virus of host adaptive immune response / metalloendopeptidase inhibitor activity / positive regulation of calcium ion-dependent exocytosis / suppression by virus of host autophagy / positive regulation of natural killer cell mediated cytotoxicity / long-term synaptic depression / Golgi to plasma membrane transport / determination of left/right symmetry / regulation of calcium-mediated signaling
Function and homology information
Specimen sourceHomo sapiens (human)
Human immunodeficiency virus 1
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.27 Å resolution
AuthorsBuffalo, C.Z. / Morris, K.L. / Hurley, J.H.
CitationJournal: Cell / Year: 2018
Title: HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.
Authors: Kyle L Morris / Cosmo Z Buffalo / Christina M Stürzel / Elena Heusinger / Frank Kirchhoff / Xuefeng Ren / James H Hurley
Abstract: The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, ...The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 25, 2018 / Release: Aug 8, 2018

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Assembly

Deposited unit
T: Bone marrow stromal antigen 2,Protein Nef
L: Bone marrow stromal antigen 2,Protein Nef
B: AP-1 complex subunit beta-1
C: ADP-ribosylation factor 1
G: AP-1 complex subunit gamma-1
H: ADP-ribosylation factor 1
M: AP-1 complex subunit mu-1
S: AP-1 complex subunit sigma-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)306,75912
Polyers305,6648
Non-polymers1,0954
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 2 types, 4 molecules TLCH

#1: Protein/peptide Bone marrow stromal antigen 2,Protein Nef / BST-2 / HM1.24 antigen / Tetherin / 3'ORF / Negative factor / F-protein


Mass: 29880.166 Da / Num. of mol.: 2
Fragment: Tetherin UNP residues 2-21, Nef UNP residues 1-206
Mutation: L164A, L165A
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human immunodeficiency virus 1
Gene: BST2, nef / Production host: Escherichia coli (E. coli) / References: UniProt: Q10589, UniProt: Q90VU7
#3: Protein/peptide ADP-ribosylation factor 1 / ARF1


Mass: 22314.250 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: ARF1 / Production host: Escherichia coli (E. coli) / References: UniProt: P84077

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AP-1 complex subunit ... , 4 types, 4 molecules BGMS

#2: Protein/peptide AP-1 complex subunit beta-1 / AP-1 transcription factor / Adaptor protein complex AP-1 subunit beta-1 / Adaptor-related protein complex 1 subunit beta-1 / Beta-1-adaptin / Beta-adaptin 1 / Clathrin assembly protein complex 1 beta large chain / Golgi adaptor HA1/AP1 adaptin beta subunit


Mass: 66152.547 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: AP1B1, ADTB1, BAM22, CLAPB2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q10567
#4: Protein/peptide AP-1 complex subunit gamma-1 / AP-1 transcription factor / Adaptor protein complex AP-1 subunit gamma-1 / Adaptor-related protein complex 1 subunit gamma-1 / Clathrin assembly protein complex 1 gamma-1 large chain / Gamma-adaptin / Gamma1-adaptin / Golgi adaptor HA1/AP1 adaptin subunit gamma-1


Mass: 68194.094 Da / Num. of mol.: 1 / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap1g1, Adtg, Clapg1 / Production host: Escherichia coli (E. coli) / References: UniProt: P22892
#5: Protein/peptide AP-1 complex subunit mu-1 / AP-1 transcription factor / AP-mu chain family member mu1A / Adaptor protein complex AP-1 subunit mu-1 / Adaptor-related protein complex 1 subunit mu-1 / Clathrin assembly protein complex 1 mu-1 medium chain 1 / Clathrin coat assembly protein AP47 / Clathrin coat-associated protein AP47 / Golgi adaptor HA1/AP1 adaptin mu-1 subunit / Mu-adaptin 1 / Mu1A-adaptin


Mass: 48606.730 Da / Num. of mol.: 1 / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap1m1, Cltnm / Production host: Escherichia coli (E. coli) / References: UniProt: P35585
#6: Protein/peptide AP-1 complex subunit sigma-3 / AP-1 transcription factor / Adaptor protein complex AP-1 subunit sigma-1C / Adaptor-related protein complex 1 subunit sigma-1C / Clathrin assembly protein complex 1 sigma-1C small chain / Golgi adaptor HA1/AP1 adaptin sigma-1C subunit / Sigma 1C subunit of AP-1 clathrin / Sigma-adaptin 1C / Sigma1C-adaptin


Mass: 18321.338 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: AP1S3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q96PC3

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Non-polymers , 2 types, 4 molecules

#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Formula: Mg / Magnesium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of the HIV-Nef tetherin cargo bound AP-1:Arf1 closed trimer monomeric subunit
Type: COMPLEX / Entity ID: 2,3,4,5,6 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: 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: 39.7 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 85592 / Symmetry type: POINT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00516873
ELECTRON MICROSCOPYf_angle_d0.84922827
ELECTRON MICROSCOPYf_dihedral_angle_d8.50210348
ELECTRON MICROSCOPYf_chiral_restr0.0532634
ELECTRON MICROSCOPYf_plane_restr0.0062890

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