Protein-Protein Interactions of the Human Iron Sulfur Cluster Biosynthesis Complex
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Iron sulfur (Fe-S) clusters are integral cofactors responsible for a number of cellular processes including electron transfer, catalyzing substrate turnover, sensing small molecules, and regulating gene expression or enzymatic activity. Elaborate multi-component systems have evolved to protect organism from the toxic effects of free iron and sulfide ions while promoting the efficient biosynthesis of these cofactors. Previously, our lab discovered the human cysteine desulfurase complex (NFS1-ISD11, named SD) and the Fe-S assembly scaffold protein ISCU2 form a low activity Fe-S assembly complex (named SDU) that can be activated by the allosteric activator frataxin (FXN) to generate the high activity SDUF Fe-S assembly complex. Importantly, mutations in FXN result in the neurodegenerative disease Friedreich’s Ataxia (FRDA), whereas mutations in ISCU2 lead to a disease characterized by myopathy with exercise intolerance. The goals of this dissertation were to provide structural details for protein-protein interactions in the human SDUF complex, contribute to understanding how FXN binds and activates the assembly complex, and define how a clinical ISCU2 variant was compromised in Fe-S assembly activity. To address these questions, a multidisciplinary approach was initiated that included anaerobic biochemistry and kinetic assays, fluorophore incorporation for anisotropy measurements, and chemical modifications coupled to mass spectrometry experiments. First, protein interfaces were probed by hydroxyl radical footprinting experiments where the SD, SDU, and SDUF complexes were exposed to different does of synchrotron radiation (generating hydroxyl radicals) and the resulting modified proteins were proteolytically digested and analyzed by MALDI mass spectrometry. These experiments revealed that ISCU2 binding to the SD complex results in a decrease in modification kinetics to for regions of ISCU2 near the N-terminus. Consistent with this assignment, kinetic assays revealed that the clinical ISCU2 variant, which has a mutation near the N-terminus, exhibits cysteine desulfurase and Fe-S assembly activities similar to native ISCU2, but compromised binding affinity to the assembly complex. Next, hydroxyl radical footprinting experiments revealed that FXN binding to the SDU complex resulted in the C-terminal α-helix of ISCU2 becoming more protected and suggested specific interactions associated with FXN activation. Next, fluorescence anisotropy experiments under different experimental conditions revealed both determinants for FXN binding and that the FRDA variant has compromised binding affinity to the SDU complex. Finally, this activation model was tested and supported by mutagenesis and binding studies that indicated residues in this C-terminal α-helix of ISCU2 interacts with residues on the β-sheet region of FXN, which are associated with FRDA. Together, these studies reveal details of the protein-protein interactions and function of the human SDUF complex that have implications for human disease.
hydroxyl radical footprinting
MALDI mass spectrometry
Levy, Michaella J (2014). Protein-Protein Interactions of the Human Iron Sulfur Cluster Biosynthesis Complex. Doctoral dissertation, Texas A & M University. Available electronically from