During FeS cluster biogenesis, ferredoxin and frataxin use overlapping binding sites on yeast cysteine desulfurase Nfs1

Marta A Uzarska,Igor Grochowina,Joanna Soldek,Marcin Jelen,Brenda Schilke,Jaroslaw Marszalek,Elizabeth A Craig,Rafal Dutkiewicz

doi:10.1016/j.jbc.2022.101570

Marta A Uzarska, Igor Grochowina + Show 6 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101570

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Abstract

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron–sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins. Nfs1 functions both as a sulfur donor for cluster assembly and as a binding platform for other proteins functioning in the process. These include not only the dedicated scaffold protein (Isu1) on which FeS clusters are synthesized but also accessory FeS cluster biogenesis proteins frataxin (Yfh1) and ferredoxin (Yah1). Yfh1 has been shown to activate cysteine desulfurase enzymatic activity, whereas Yah1 supplies electrons for the persulfide reduction. While Yfh1 interaction with Nfs1 is well understood, the Yah1–Nfs1 interaction is not. Here, based on the results of biochemical experiments involving purified WT and variant proteins, we report that in Saccharomyces cerevisiae, Yah1 and Yfh1 share an evolutionary conserved interaction site on Nfs1. Consistent with this notion, Yah1 and Yfh1 can each displace the other from Nfs1 but are inefficient competitors when a variant with an altered interaction site is used. Thus, the binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system. Our findings are consistent with the generally accepted scenario that the mitochondrial FeS cluster assembly system was inherited from bacterial ancestors of mitochondria.

Highlights

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron–sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins
The binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system
biolayer interferometry (BLI) sensors were loaded with Yah1GST or GST as indicated

Summary

Results

With a goal of analyzing the interaction of Yah with NIA and the effect of the presence of Isu (Fig. 1A), we used. NIA was rapidly recruited to the sensors carrying Yah1GST (Fig. 1F) with equilibrium dissociation constant (KD) values in the micromolar range (Fig. S5), consistent with the results obtained using the pull-down assay Because both binding assays described previously utilized the Yah1GST fusion protein, we tested whether the presence of the GST tag affects the interaction of Yah with the NIA complex. Yah was a less efficient competitor than Yfh, as a 75 μM concentration of Yah was needed to reduce the amounts of Nfs, Isd, and Isu1Ct pulled down with Yfh1GST to 35% of the control value measured in the absence of Yah1 This result was further verified using the BLI assay (Fig. 2G). The computationally predicted mode of Yah1–NIA–Isu interaction is consistent with our biochemical results and very similar to the mode of bacterial Fdx–IscS–IscU interaction [27]

Discussion

Experimental procedures

Methods