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Abstract
Iron-sulfur clusters are essential protein cofactors synthesized in human mitochondria by an NFS1-ISD11-ACP-ISCU2-FXN assembly complex. Surprisingly, researchers have discovered three distinct quaternary structures for cysteine desulfurase subcomplexes, which display similar interactions between NFS1-ISD11-ACP protomeric units but distinct dimeric interfaces between the protomers. Although the role of these different architectures is unclear, possible functions include regulating activity and promoting the biosynthesis of distinct sulfur-containing biomolecules. Here, crystallography, native ion-mobility mass spectrometry, and chromatography methods reveal the Fe-S assembly subcomplex exists as an equilibrium mixture of these different quaternary structures. Our results suggest Friedreich's ataxia (FRDA) protein frataxin (FXN) functions as a "molecular lock" and shifts the equilibrium towards one of the architectures to stimulate the cysteine desulfurase activity and promote iron-sulfur cluster biosynthesis. An NFS1-designed variant similarly shifts the equilibrium and partially replaces FXN in activating the complex. These results suggest that eukaryotic cysteine desulfurases are unusual members of the morpheein class of enzymes that control their activity through their oligomeric state. Overall, the findings support architectural switching as a regulatory mechanism linked to FXN activation of the human Fe-S cluster biosynthetic complex and provide new opportunities for therapeutic interventions of the fatal neurodegenerative disease FRDA.
Supplementary materials
Title
Supplementary Matrials
Description
The supplementary materials contain SAXS parameters under low (Table S1) and high salt conditions (Table S2), crystallographic space groups for AI and TB-prepared complexes (Table S3), kinetic exchange data with native mass spectrometry (Table S4), collisional cross-section experimental and calculated values complexes probed by ion mobility mass spectrometry experiments (Table S5), a summary of the functional and bioanalytical and biophysical analysis of the different architectures (Table S6), SAXS data collection parameters (Table S7), and calculated and measured masses for SDAec complexes (Table S8). The supplementary materials also contain figures that describe structural overlays and models, kinetic analysis, SAXS scattering curve overlays, crystallization results, native MS spectra from the mixed isotope exchange experiments, cation exchange results, additional ion mobility mass spectrometry experiments of the complexes as a function of temperature, batch, and the presence of the his-tag.
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