Histidine 103 in Fra2 Is an Iron-Sulfur Cluster Ligand in the [2Fe-2S] Fra2-Grx3 Complex and Is Required for in Vivo Iron Signaling in Yeast

Haoran Li,Daphne T Mapolelo,Nin N Dingra,Greg Keller,Pamela J Riggs-Gelasco,Dennis R Winge,Michael K Johnson,Caryn E Outten

doi:10.1074/jbc.m110.184176

Abstract

The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. To further characterize this unusual Fe-S-binding complex, site-directed mutagenesis was used to identify specific residues in Fra2 that influence Fe-S cluster binding and regulation of Aft1 activity in vivo. Here, we present spectroscopic evidence that His-103 in Fra2 is an Fe-S cluster ligand in the Fra2-Grx3 complex. Replacement of this residue does not abolish Fe-S cluster binding, but it does lead to a change in cluster coordination and destabilization of the [2Fe-2S] cluster. In vivo genetic studies further confirm that Fra2 His-103 is critical for control of Aft1 activity in response to the cellular iron status. Using CD spectroscopy, we find that ∼1 mol eq of apo-Fra2 binds tightly to the [2Fe-2S] Grx3 homodimer to form the [2Fe-2S] Fra2-Grx3 heterodimer, suggesting a mechanism for formation of the [2Fe-2S] Fra2-Grx3 heterodimer in vivo. Taken together, these results demonstrate that the histidine coordination and stability of the [2Fe-2S] cluster in the Fra2-Grx3 complex are essential for iron regulation in yeast.

Highlights

The iron-responsive transcriptional activator Aft1 and its paralogue Aft2 [1,2,3]
Grx3 mutagenesis studies demonstrate that Cys-176, located in the conserved Grx-like domain active site (CGFS), and residues in the GSH binding pocket (Trp214/Pro-215) are required for heterodimer formation and cluster binding
Because the spectroscopic data suggest that both His and Cys residues play a role in cluster binding [9], yeast Fra2 was modeled on the structure of its mouse homologue (BolA2) to identify well conserved Cys/His residues located within close proximity to each other

Summary

Introduction

The iron-responsive transcriptional activator Aft and its paralogue Aft2 [1,2,3]. Under iron-replete conditions, Aft is mainly cytosolic, and under iron-deplete conditions, Aft accumulates in the nucleus where it activates genes involved in iron uptake and transport, collectively known as the iron regulon [4]. The specific role of the Fra-Grx complex and the precise mechanism of iron-dependent inhibition of Aft1/2 activity are still unclear To better understand this signaling pathway, we recently characterized the molecular interactions between Fra and Grx3/4 [9]. Grx mutagenesis studies demonstrate that Cys-176, located in the conserved Grx-like domain active site (CGFS), and residues in the GSH binding pocket (Trp214/Pro-215) are required for heterodimer formation and cluster binding. These same residues are indispensable for iron-dependent inhibition of Aft activity in vivo [6], suggesting that there is a direct link between the formation of the [2Fe-2S] Fra2-Grx3/4 complex and their in vivo role in iron signaling. These results provide a more detailed picture of the Fra2-Grx interaction and support a model in which Fra acts as an adaptor protein converting Grx from an Fe-S scaffold/delivery protein with a relatively labile cluster to an iron sensor with a stable cluster

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Conclusion