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- PDB-6vcd: Cryo-EM structure of IRP2-FBXL5-SKP1 complex -

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Basic information

Entry
Database: PDB / ID: 6vcd
TitleCryo-EM structure of IRP2-FBXL5-SKP1 complex
Components
  • F-box/LRR-repeat protein 5
  • Iron-responsive element binding protein 2, isoform CRA_a
  • S-phase kinase-associated protein 1
KeywordsLIGASE / E3 ligase / [2Fe-2S] cluster / Iron metabolism
Function / homology
Function and homology information


aconitate hydratase activity / iron-responsive element binding / : / F-box domain binding / citrate metabolic process / PcG protein complex / protoporphyrinogen IX biosynthetic process / Cul7-RING ubiquitin ligase complex / positive regulation of ubiquitin protein ligase activity / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling ...aconitate hydratase activity / iron-responsive element binding / : / F-box domain binding / citrate metabolic process / PcG protein complex / protoporphyrinogen IX biosynthetic process / Cul7-RING ubiquitin ligase complex / positive regulation of ubiquitin protein ligase activity / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / maintenance of protein location in nucleus / erythrocyte homeostasis / intestinal absorption / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / SCF ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / Prolactin receptor signaling / protein monoubiquitination / Association of TriC/CCT with target proteins during biosynthesis / cullin family protein binding / ubiquitin-like ligase-substrate adaptor activity / protein K48-linked ubiquitination / Nuclear events stimulated by ALK signaling in cancer / ubiquitin ligase complex / translation repressor activity / tricarboxylic acid cycle / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / osteoclast differentiation / post-embryonic development / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / molecular function activator activity / Vpu mediated degradation of CD4 / Dectin-1 mediated noncanonical NF-kB signaling / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Negative regulation of NOTCH4 signaling / Iron uptake and transport / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Degradation of beta-catenin by the destruction complex / 2 iron, 2 sulfur cluster binding / NOTCH1 Intracellular Domain Regulates Transcription / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / beta-catenin binding / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / multicellular organismal-level iron ion homeostasis / FCERI mediated NF-kB activation / Interleukin-1 signaling / protein polyubiquitination / Orc1 removal from chromatin / ubiquitin-protein transferase activity / positive regulation of protein catabolic process / Regulation of RUNX2 expression and activity / Cyclin D associated events in G1 / Regulation of PLK1 Activity at G2/M Transition / Antigen processing: Ubiquitination & Proteasome degradation / Circadian Clock / Downstream TCR signaling / Neddylation / 4 iron, 4 sulfur cluster binding / iron ion transport / proteasome-mediated ubiquitin-dependent protein catabolic process / intracellular iron ion homeostasis / protein ubiquitination / chromatin remodeling / iron ion binding / protein domain specific binding / centrosome / perinuclear region of cytoplasm / mitochondrion / RNA binding / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Iron regulatory protein 2 / Aconitase/Iron-responsive element-binding protein 2 / Aconitase A, swivel domain / FBXL5-like, hemerythrin-like domain / Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/alpha domain / Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/alpha, subdomain 1/3 / Aconitase family, 4Fe-4S cluster binding site / Aconitase, iron-sulfur domain / Aconitase family (aconitate hydratase) / Aconitase family signature 1. ...Iron regulatory protein 2 / Aconitase/Iron-responsive element-binding protein 2 / Aconitase A, swivel domain / FBXL5-like, hemerythrin-like domain / Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/alpha domain / Aconitase/3-isopropylmalate dehydratase large subunit, alpha/beta/alpha, subdomain 1/3 / Aconitase family, 4Fe-4S cluster binding site / Aconitase, iron-sulfur domain / Aconitase family (aconitate hydratase) / Aconitase family signature 1. / Aconitase family signature 2. / Aconitase A/isopropylmalate dehydratase small subunit, swivel domain / Aconitase C-terminal domain / Aconitase/3-isopropylmalate dehydratase, swivel / Hemerythrin-like / Hemerythrin HHE cation binding domain / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / F-box domain profile. / F-box-like domain superfamily / F-box-like / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / F-box domain / Leucine Rich repeat / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
FE2/S2 (INORGANIC) CLUSTER / Iron-responsive element-binding protein 2 / Iron-responsive element-binding protein 2 / S-phase kinase-associated protein 1 / F-box/LRR-repeat protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsWang, H. / Shi, H. / Zheng, N.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute United States
CitationJournal: Mol Cell / Year: 2020
Title: FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.
Authors: Hui Wang / Hui Shi / Malini Rajan / Elizabeth R Canarie / Seoyeon Hong / Daniele Simoneschi / Michele Pagano / Matthew F Bush / Stefan Stoll / Elizabeth A Leibold / Ning Zheng /
Abstract: Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between ...Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.
History
DepositionDec 20, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 5, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Iron-responsive element binding protein 2, isoform CRA_a
B: F-box/LRR-repeat protein 5
C: S-phase kinase-associated protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)178,8544
Polymers178,6783
Non-polymers1761
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4820 Å2
ΔGint-41 kcal/mol
Surface area43190 Å2

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Components

#1: Protein Iron-responsive element binding protein 2, isoform CRA_a / IRP2


Mass: 105165.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IREB2, hCG_38938 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: D3DW85, UniProt: P48200*PLUS
#2: Protein F-box/LRR-repeat protein 5 / FBXL5 / F-box and leucine-rich repeat protein 5 / F-box protein FBL4/FBL5 / p45SKP2-like protein


Mass: 54832.441 Da / Num. of mol.: 1 / Fragment: UNP residues 183-674
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FBXL5, FBL4, FBL5, FLR1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UKA1
#3: Protein S-phase kinase-associated protein 1 / / SKP1 / Cyclin-A/CDK2-associated protein p19 / p19A / Organ of Corti protein 2 / OCP-2 / Organ of ...SKP1 / Cyclin-A/CDK2-associated protein p19 / p19A / Organ of Corti protein 2 / OCP-2 / Organ of Corti protein II / OCP-II / RNA polymerase II elongation factor-like protein / SIII / Transcription elongation factor B polypeptide 1-like / p19skp1


Mass: 18679.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SKP1, EMC19, OCP2, SKP1A, TCEB1L / Production host: Escherichia coli (E. coli) / References: UniProt: P63208
#4: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster


Mass: 175.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe2S2 / 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: IRP2-FBLX5-SKP1 complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 73.8 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.15.2_3472refinement
PHENIX1.15.2_3472refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 955060 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00448253
ELECTRON MICROSCOPYf_angle_d0.617311180
ELECTRON MICROSCOPYf_chiral_restr0.04421275
ELECTRON MICROSCOPYf_plane_restr0.00461424
ELECTRON MICROSCOPYf_dihedral_angle_d9.67624934

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