Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism

Abstract

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.

Fig. 1

SDAUF-Zn²⁺ structure and FXN-NFS1 interactions. a. Cryo-EM density of SDAUF-Zn²⁺ structure (NFS1/NFS1’ slate, ISD11/ISD11’ magenta, ACP/ACP’ light green, ISCU/ISCU’, Cyan, FXN/FXN’ orange). b. Cartoon representation of SDAUF-Zn²⁺ complex. 4′-phosphopantetheine acyl chain (8Q1, yellow), Zn²⁺ ion (ZN, red) and pyridoxal 5′-phosphate (PLP, wheat) are shown as sticks/spheres. c. Scattering data from a sample of SDAUF used in cryo-EM was collected (black points) and fit to the theoretical SAXS profile back-calculated from the SDAUF-Zn²⁺ cryo-EM structure (red line) with χ² = 1.98. d. The ab initio envelope calculated from SAXS data for the SDAUF sample was superimposed with the cryo-EM structure e. In the SDAUF complex, each FXN binds to a cavity formed by interface of NFS1 homodimer and one ISCU. FXN is shown as orange cartoon, while other subunits of the complex are shown as surface representation (colored by electrostatic potential). Yellow dotted lines denote protein boundaries. Source data for panel c are provided as a Source Data file

Fig. 2

FXN-ISCU interactions and FXN mutagenesis. a. Interactions of FXN (orange) with both NFS1 subunits (NFS1, light slate; NFS1′, blue). Residues studied by site-directed mutagenesis are asterisked. Dashed lines denote potential hydrogen bonds. b. Interface of FXN β-sheet with ISCU LPPVK-region and NFS1 Cys-loop. c. Interface of FXN with ISCU Ala-loop, LPPVK-region, and Zn²⁺ ion (sphere). Inset shows viewpoints of panels b and c within SDAUF-Zn²⁺ complex

Fig. 3

Comparing structures with and without FXN. a. Same view of SDAUF-Zn²⁺ as Fig. 2c, superimposed with ISCU subunits from SDAU-Zn²⁺ (5WLW) and SDAU (5WKP) structures. NFS1 Cys-loops from SDAUF-Zn²⁺ and SDAU-Zn²⁺ are shown. b. Sequence alignment of FXN region containing Pro163 (human numbering) and the ISCU L₁₃₁PPVKLHCSM₁₄₀ loop. Sequences shown include FXN and ISCU orthologues from human (Uniprot ID Q16595 and Q9H1K1, respectively), S. cerevisiae (Q07540, Q03020), E. coli (P27838, P0ACD4), H. influenzae (P71358, Q57074) and P. aeruginosa (Q9HTS5, Q9HXI9) c. ISCU L₁₃₁PPVKLHCSM₁₄₀ loop from the superimposed structures, adjacent to FXN Pro163 (orange). Human ISCU encodes Met at residue 140 (cyan stick), while in SDAU-Zn²⁺ and SDAU structures Met was substituted to Ile (white and pink sticks respectively). In both panels a and c, FXN mutagenesis residues and ISCU Met140 are asterisked, and Zn²⁺ ions are shown as spheres. Dashed lines denote potential hydrogen bonds or Zn²⁺ coordination

Fig. 4

Proposed mechanistic features of FXN activation on NFS1 and ISCU. Schematic of the NFS1 Cys-loop trajectory, mediated by FXN-induced conformational changes on ISCU in the LPPVK region. Subunits of NFS1, ISCU and FXN are colored violet, cyan and orange respectively. Key residues discussed in the text are shown as sticks. The Cys-loop from NFS1 is shown as coil, and the LPPVK region Inset shows the orientation of complex from which the cartoon representation is derived