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- PDB-8pk8: Structure of the human mitochondrial iron-sulfur cluster biosynth... -

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Basic information

Entry
Database: PDB / ID: 8pk8
TitleStructure of the human mitochondrial iron-sulfur cluster biosynthesis complex during persulfide transfer (persulfide on ISCU2)
Components
  • Acyl carrier protein
  • Cysteine desulfurase
  • Frataxin mature form
  • Iron-sulfur cluster assembly enzyme ISCU
  • LYR motif-containing protein 4
KeywordsTRANSFERASE / cysteine desulfurase / FeS biosynthesis / FeS biogenesis / mitochondria / Friedreich's ataxia / persulfide / frataxin
Function / homology
Function and homology information


positive regulation of lyase activity / iron-sulfur cluster chaperone activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / proprioception / Molybdenum cofactor biosynthesis / L-cysteine desulfurase complex / [4Fe-4S] cluster assembly / Mitochondrial iron-sulfur cluster biogenesis / sulfur carrier activity ...positive regulation of lyase activity / iron-sulfur cluster chaperone activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / proprioception / Molybdenum cofactor biosynthesis / L-cysteine desulfurase complex / [4Fe-4S] cluster assembly / Mitochondrial iron-sulfur cluster biogenesis / sulfur carrier activity / Complex III assembly / iron chaperone activity / positive regulation of mitochondrial electron transport, NADH to ubiquinone / Maturation of TCA enzymes and regulation of TCA cycle / cysteine desulfurase / cysteine desulfurase activity / negative regulation of organ growth / Mo-molybdopterin cofactor biosynthetic process / mitochondrial respiratory chain complex III assembly / embryo development ending in birth or egg hatching / Mitochondrial protein import / iron-sulfur cluster assembly complex / mitochondrial [2Fe-2S] assembly complex / oxidative phosphorylation / response to iron ion / heme biosynthetic process / adult walking behavior / [2Fe-2S] cluster assembly / negative regulation of multicellular organism growth / organ growth / iron-sulfur cluster assembly / lipid A biosynthetic process / muscle cell cellular homeostasis / lipid biosynthetic process / ferroxidase / negative regulation of release of cytochrome c from mitochondria / acyl binding / acyl carrier activity / protein autoprocessing / ferroxidase activity / iron-sulfur cluster binding / phosphopantetheine binding / ferric iron binding / protein maturation / enzyme activator activity / iron ion transport / ferrous iron binding / 2 iron, 2 sulfur cluster binding / cellular response to hydrogen peroxide / fatty acid biosynthetic process / pyridoxal phosphate binding / Maturation of replicase proteins / molecular adaptor activity / intracellular iron ion homeostasis / nuclear body / iron ion binding / mitochondrial matrix / response to xenobiotic stimulus / centrosome / lipid binding / negative regulation of apoptotic process / structural molecule activity / protein homodimerization activity / mitochondrion / zinc ion binding / nucleoplasm / metal ion binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
LYRM4, LYR domain / : / Frataxin / ISC system FeS cluster assembly, IscU scaffold / Frataxin/CyaY / Frataxin conserved site / Frataxin-like domain / Frataxin family signature. / Frataxin family profile. / Frataxin-like domain ...LYRM4, LYR domain / : / Frataxin / ISC system FeS cluster assembly, IscU scaffold / Frataxin/CyaY / Frataxin conserved site / Frataxin-like domain / Frataxin family signature. / Frataxin family profile. / Frataxin-like domain / Cysteine desulfurase IscS / NIF system FeS cluster assembly, NifU, N-terminal / NifU-like N terminal domain / Frataxin/CyaY superfamily / Cysteine desulfurase / Aminotransferase class-V, pyridoxal-phosphate binding site / Aminotransferases class-V pyridoxal-phosphate attachment site. / Aminotransferase class V domain / Aminotransferase class-V / Complex 1 LYR protein domain / Complex 1 protein (LYR family) / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Pyridoxal phosphate-dependent transferase, small domain / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
Chem-8Q1 / : / PYRIDOXAL-5'-PHOSPHATE / Acyl carrier protein / Frataxin, mitochondrial / Iron-sulfur cluster assembly enzyme ISCU / LYR motif-containing protein 4 / Cysteine desulfurase
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsSteinhilper, R. / Murphy, B.J.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2024
Title: Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.
Authors: Vinzent Schulz / Ralf Steinhilper / Jonathan Oltmanns / Sven-A Freibert / Nils Krapoth / Uwe Linne / Sonja Welsch / Maren H Hoock / Volker Schünemann / Bonnie J Murphy / Roland Lill /
Abstract: Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11- ...Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381 to Cys138 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.
History
DepositionJun 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cysteine desulfurase
B: LYR motif-containing protein 4
C: Acyl carrier protein
D: Iron-sulfur cluster assembly enzyme ISCU
E: Frataxin mature form
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,2008
Polymers97,3565
Non-polymers8443
Water2,576143
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 5 molecules ABCDE

#1: Protein Cysteine desulfurase


Mass: 44937.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NFS1, NIFS, HUSSY-08 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9Y697, cysteine desulfurase
#2: Protein LYR motif-containing protein 4


Mass: 13502.373 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LYRM4, C6orf149, ISD11, CGI-203 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9HD34
#3: Protein Acyl carrier protein / ACP / Cytosolic-activating factor / CAF / Fatty acid synthase acyl carrier protein


Mass: 8645.460 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A6A8
#4: Protein Iron-sulfur cluster assembly enzyme ISCU / NifU-like N-terminal domain-containing protein / NifU-like protein


Mass: 15716.184 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ISCU, NIFUN / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9H1K1
#5: Protein Frataxin mature form / Frataxin(81-210) / m81-FXN


Mass: 14554.985 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FXN, FRDA, X25 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q16595

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Non-polymers , 4 types, 146 molecules

#6: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H10NO6P / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-8Q1 / S-[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] dodecanethioate / S-dodecanoyl-4'-phosphopantetheine


Mass: 540.651 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H45N2O8PS / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-FE2 / FE (II) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 143 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex during persulfide transfer
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
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
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
7Coot0.9model fitting
12RELION3.1.33D reconstruction
19PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 507565 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6NZU

6nzu


Accession code: 6NZU / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00512805
ELECTRON MICROSCOPYf_angle_d0.73617298
ELECTRON MICROSCOPYf_dihedral_angle_d5.3421805
ELECTRON MICROSCOPYf_chiral_restr0.0481954
ELECTRON MICROSCOPYf_plane_restr0.0072218

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