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.
Highlights
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
FXN binding to the SDAU complex is dynamic, yielding low-μM dissociation constants (Kd) by bio-layer interferometry (BLI) (Supplementary Fig. 1a-c), presenting challenges to isolate the SDAUF complex intact with all 5 components in proper stoichiometry
We co-expressed in E. coli a plasmid containing His6ISD11-NFS1-ISCU, with a plasmid containing His6-FXN (Supplementary Fig. 2c)
Summary
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. Upon co-expression with the plasmid containing His6-ISD11-NFS1-ISCU, we observed a heterogeneous complex containing an approximately equimolar mixture of the desired human ACP and contaminating ACPec (Supplementary Fig. 2d).
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