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bioRxiv · 10.1101/2020.10.06.326603

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Patra, S.·Lin, C.-W.·Ghosh, M. K.·Havens, S. M.·Cory, S. A.·Russell, D. H.·Barondeau, D. P.
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Abstract

Iron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share [~]60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreichs ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.

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Patra, S., Lin, C.-W., Ghosh, M. K., Havens, S. M., Cory, S. A., Russell, D. H., Barondeau, D. P.. 2020-10-06. Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model. https://doi.org/10.1101/2020.10.06.326603

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