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.

Highlights

  • Fe-S clusters are highly versatile cofactors that prokaryotic and eukaryotic cells utilize in many different proteins for a vari

  • These previous data together with the high degree of conservation between prokaryotic and mitochondrial Fe-S cluster assembly systems [2, 3, 34] led us to hypothesize that simultaneous co-expression of FXN42–210, NFS1, ISD11, and ISCU in E. coli might mimic the mitochondrial environment and yield similar complexes in sufficient quantities to enable structural studies

  • At the end of the protein expression phase, size-exclusion chromatography and SDS-PAGE analysis of the total bacterial cell extract revealed that similar levels of FXN42–210, ISCU, and ISD11 were co-eluted with NFS1, which was present in large excess relative to the other three proteins

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Summary

Results

Isolation and Biochemical Characterization of Human Fe-S Cluster Assembly Complex—We previously reconstituted a functional [FXN42–210]n1⁄7[NFS1]n1⁄7[ISD11]n1⁄7[ISCU]n complex in vitro from independently expressed and purified FXN42–210 oligomer, [NFS1]1⁄7[ISD11] sub-complex, and ISCU [7]. EM Single Particle Reconstruction of the [FXN42–210]n1⁄7 [NFS1]n1⁄7[ISD11]n1⁄7[ISCU]n Complex—Uranyl acetate staining of aliquots from pooled fractions 56 – 60 revealed roughly globular shapes with a diameter of 15.3 Ϯ 0.7 nm (average of 351 particles) (Fig. 2A and not shown), consistent with the Rh measured by dynamic light scattering and the behavior of the complex in size-exclusion chromatography. These data together suggested we were dealing with an overall homogeneous population of roughly globular particles.

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