Architecture of the human mitochondrial iron-sulfur cluster assembly machinery

Oleksandr Gakh, Wasantha Ranatunga, Douglas Y. Smith, Eva Christina Ahlgren, Salam Al-Karadaghi, James R. Thompson, Grazia Isaya

Research output: Contribution to journalArticle

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Abstract

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42-210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42-210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42-210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42-210 to ISCU.

Original languageEnglish (US)
Pages (from-to)21296-21321
Number of pages26
JournalJournal of Biological Chemistry
Volume291
Issue number40
DOIs
StatePublished - Sep 30 2016

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sulofenur
Sulfur
Machinery
Iron
Stoichiometry
Proteins
Negative Staining
Mitochondria
Molecular Dynamics Simulation
Macromolecules
Hydrophobic and Hydrophilic Interactions
Scaffolds
Escherichia coli
Molecular dynamics
Hydrogen
Bacteria
Hydrogen bonds
Salts
Organizations

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Gakh, O., Ranatunga, W., Smith, D. Y., Ahlgren, E. C., Al-Karadaghi, S., Thompson, J. R., & Isaya, G. (2016). Architecture of the human mitochondrial iron-sulfur cluster assembly machinery. Journal of Biological Chemistry, 291(40), 21296-21321. https://doi.org/10.1074/jbc.M116.738542

Architecture of the human mitochondrial iron-sulfur cluster assembly machinery. / Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y.; Ahlgren, Eva Christina; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia.

In: Journal of Biological Chemistry, Vol. 291, No. 40, 30.09.2016, p. 21296-21321.

Research output: Contribution to journalArticle

Gakh, O, Ranatunga, W, Smith, DY, Ahlgren, EC, Al-Karadaghi, S, Thompson, JR & Isaya, G 2016, 'Architecture of the human mitochondrial iron-sulfur cluster assembly machinery', Journal of Biological Chemistry, vol. 291, no. 40, pp. 21296-21321. https://doi.org/10.1074/jbc.M116.738542
Gakh O, Ranatunga W, Smith DY, Ahlgren EC, Al-Karadaghi S, Thompson JR et al. Architecture of the human mitochondrial iron-sulfur cluster assembly machinery. Journal of Biological Chemistry. 2016 Sep 30;291(40):21296-21321. https://doi.org/10.1074/jbc.M116.738542
Gakh, Oleksandr ; Ranatunga, Wasantha ; Smith, Douglas Y. ; Ahlgren, Eva Christina ; Al-Karadaghi, Salam ; Thompson, James R. ; Isaya, Grazia. / Architecture of the human mitochondrial iron-sulfur cluster assembly machinery. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 40. pp. 21296-21321.
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