Iron-sulfur clusters are involved in post-translational arginylation

Abstract

Eukaryotic arginylation is an essential post-translational modification that both modulates protein stability and regulates protein half-life through the N-degron pathway. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite its conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we have discovered that ATE1s bind a previously unknown iron-sulfur cluster that is conserved across evolution. We have extensively characterized the nature of this iron-sulfur cluster, and we show that the presence of the iron-sulfur cluster is linked to alterations in arginylation efficacy. Finally, we demonstrate that the ATE1 iron-sulfur cluster is oxygen sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Thus, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

The structure of arginyltransferase 1 (ATE1)

“…Based on our previous work, we have demonstrated that the [Fe-S] cluster-binding site is composed of Cys 23 and Cys 26 (part of a conserved CGYC motif) and Cys 94 /Cys 95 (part of a conserved CC motif). 32 While we have yet been able to crystallize the holo (cluster-replete) form of the protein, the AlphaFold model of ScATE1 supports co-localization of all four Cys residues in spatial proximity. Our apo structure reveals that the CGYC motif is part of a dynamic and disordered loop stretching from residues 15-33 (Supplementary Figure 7), and we speculate that this region becomes ordered in the presence of the [Fe-S] cluster.…”

“…For example, our recent results have shown that ATE1 is multifaceted by also being an [Fe-S] cluster-binding enzyme. We posited that the presence of this cluster appears to function as an O 2 -dependent regulator to control post-translational arginylation in response to oxidative stress 32 , which is an important aspect of ATE1 function. While our new structure is apo (lacks the [Fe-S] cluster), it is tempting to speculate the mechanistic connection between cluster-binding in the N-terminal regulatory domain and alterations that could occur at the GNAT fold, and a metal-bound structure would significantly increase our understanding of this regulatory paradigm.…”

“…However, we were able to visualize the partial location of the [Fe-S] cluster-binding site in the N-terminal domain (Fig. 3b), which we have previously shown to be important for the regulation of arginylation 32 . Based on our previous work, we have demonstrated that the [Fe-S] cluster-binding site is composed of Cys 23 and Cys 26 (part of a conserved CGYC motif) and Cys 94 /Cys 95 (part of a conserved CC motif).…”

“…An additional complication to the determination of ATE1 structure is our recent discovery that ATE1s are O 2 -sensitive, [Fe-S] cluster-binding enzymes. 32 While we mapped the binding of this [Fe-S] cluster to the N-terminal regulatory domain of ATE1, demonstrated its effects on in vitro arginylation, and showed its evolutionary conservation, we noted that heterologous expression of ATE1 in Escherichia coli could result in heterogeneous loading of the [Fe-S] cluster if not precisely controlled. 32 This heterogeneity could hamper the formation of sufficient homogeneous protein for structural characterization.…”

“…32 While we mapped the binding of this [Fe-S] cluster to the N-terminal regulatory domain of ATE1, demonstrated its effects on in vitro arginylation, and showed its evolutionary conservation, we noted that heterologous expression of ATE1 in Escherichia coli could result in heterogeneous loading of the [Fe-S] cluster if not precisely controlled. 32 This heterogeneity could hamper the formation of sufficient homogeneous protein for structural characterization. Additionally, as ATE1 itself can be prone to aggregation, reacts with multiple protein substrates, and uses Arg tRNA Arg as a co-substrate, 13 it is perhaps unsurprising that structural insight into this enzyme has been slow.…”