title: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery.
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]24complex, 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]24sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42-210trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2sub-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 FXN42-210to ISCU.
authors: From the Departments of Pediatric and Adolescent Medicine and Biochemistry Molecular Biology, Mayo Clinic Children's Research Center, and.
authors: Oleksandr
authors: O
authors: Gakh
authors: From the Departments of Pediatric and Adolescent Medicine and Biochemistry Molecular Biology, Mayo Clinic Children's Research Center, and.
authors: Wasantha
authors: W
authors: Ranatunga
authors: From the Departments of Pediatric and Adolescent Medicine and Biochemistry Molecular Biology, Mayo Clinic Children's Research Center, and.
authors: Douglas Y
authors: DY
authors: Smith
authors: the Center for Molecular Protein Science, Institute for Chemistry and Chemical Engineering, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden.
authors: Eva-Christina
authors: EC
authors: Ahlgren
authors: the Center for Molecular Protein Science, Institute for Chemistry and Chemical Engineering, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden.
authors: Salam
authors: S
authors: Al-Karadaghi
authors: Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905 and.
authors: James R
authors: JR
authors: Thompson
authors: From the Departments of Pediatric and Adolescent Medicine and Biochemistry Molecular Biology, Mayo Clinic Children's Research Center, and isaya@mayo.edu.
authors: Grazia
authors: G
authors: Isaya
mesh_terms: D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D035925:Iron-Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000096923:Frataxin
chemical_list: D013437:Carbon-Sulfur Lyases; D033862:Iron-Binding Proteins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D000096923:Frataxin; C506160:ISCU protein, human; C544445:LYRM4 protein, human; C513492:NFS1 protein, human
keywords: Friedreich ataxia; frataxin; iron-sulfur protein; mitochondria; protein complex