Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2

Abstract

The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn(2+)-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe(2+)) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity. Furthermore, we demonstrate that the [2Fe-2S]-bridged heterodimer formed between glutaredoxin-3 and the BolA-like protein Fe repressor of activation-2 transfers a [2Fe-2S] cluster to Aft2 that facilitates Aft2 dimerization. Previous in vivo findings strongly support the [2Fe-2S] cluster-induced dimerization model; however, given the available evidence, Fe(2+)-induced Aft2 dimerization cannot be completely ruled out as an alternative Aft2 inhibition mechanism. Taken together, these data provide insight into the molecular mechanism for iron-dependent transcriptional regulation of Aft2 and highlight the key role of Fe-S clusters as cellular iron signals.

Keywords: Fra2; Grx3; iron signaling; iron–sulfur cluster; yeast.

Fig. 1.

(A) Structure of the Aft2-DNA complex. DNA is colored orange, Aft2 green, and a zinc(II) ion purple. (B) Sequence alignment of the homologous N termini of Aft2 and Aft1. Identical residues are lettered and similar residues are marked “+.” The four residues coordinating zinc(II) are highlighted in blue and the iron-sensing cysteines in orange. Secondary structural elements as determined by the crystal structure are indicated above the sequence: green arrows for β-strands and purple tubes for α-helices. Solid black lines represent loops, and dashed lines disordered residues not modeled in the structure.

Fig. 2.

Aft2 interactions with DNA. (A) Structure of the WRKY-GCM1 domain in Aft2. The zinc(II) ion is shown as a purple sphere and the metal-coordinating residues as blue sticks. The proper folding of this domain is essential to the integrity of the protein. (B) Schematic representation of Aft2-DNA contacts. (C) Close-up of protein residues within the WRKY-GCM1 domain making specific contacts with bases in the Fe-RE.

Fig. 3.

Dimerization of Aft2 in the presence of Fe²⁺ or [2Fe-2S] cluster. (A) Molecular mass of metal-loaded Aft2 determined by analytical ultracentrifugation. (B) Radii of gyration of metal-loaded Aft2 determined by SAXS. (C) Normalized electron-pair distribution function profiles of Aft2 loaded with various metals and metal species.

Fig. 4.

Interaction of Aft2 with [2Fe-2S]-Fra2-Grx3. (A, Upper) Gel-filtration chromatograms of Aft2 (blue), [2Fe-2S]-Fra2-Grx3 (red), and Aft2 plus [2Fe-2S]-Fra2-Grx3 (green). The elution positions of molecular mass standards used for column calibration are shown as dotted lines. (A, Lower) SDS/PAGE analysis of the fractions collected. Note: Fra2 typically runs as two bands on the gel (18). (B) Titration of [2Fe-2S]-Fra2-Grx3 (red line) with 0.25- to 5-fold excess Aft2 (black lines) monitored by UV-visible CD spectroscopy. Arrows indicate the direction of intensity changes with increasing Aft2 added. Blue line is 5:1 [Aft2]:[2Fe-2S] ratio. (Inset) Difference in CD intensity between 400 and 472 nm with increasing Aft2:[2Fe-2S] ratios. The dotted line highlights the 2.5:1 binding stoichiometry between Aft2 and [2Fe-2S]-Fra2-Grx3.

Fig. 5.

[2Fe-2S] cluster transfer from Fra2-Grx3 to Aft2. (A) UV-visible absorption (Upper) and CD (Lower) spectra of heparin column flow-through (red dotted line) and eluate (blue dotted line), compared with as-purified [2Fe-2S]-Fra2-Grx3 (red line) and Aft2 (blue line). ε and Δε values are normalized to Fra2-Grx3 heterodimer or Aft2 homodimer concentrations. (Inset) SDS/PAGE analysis of heparin-affinity chromatography fractions of Aft2 or [2Fe-2S]-Fra2-Grx3 alone or a 2:1 mixture of Aft2 to [2Fe-2S]-Fra2-Grx3. E, eluate; F-T, flow-through. (B) Fe-S cluster quantification in F-T and E after separation by heparin-affinity chromatography. (C, Upper) Gel-filtration chromatograms of as-purified Aft2 and eluate of [2Fe-2S] Fra2-Grx3 plus Aft2 after heparin separation. (C, Lower) SDS/PAGE analysis of the fractions collected.

Fig. 6.

Proposed model for iron regulation via Aft1 and Aft2 under iron replete conditions. During conditions of iron sufficiency, Fe–S clusters are synthesized in mitochondria via integration of iron, sulfur, and redox control pathways. An unknown substrate produced by the mitochondrial Fe-S cluster biogenesis machinery is exported to the cytosol by the transporter Atm1. GSH is also required for export of this signal. Grx3 and Grx4, which form GSH-ligated, Fe-S–bridged homodimers, are proposed to form heterodimers with Fra2 to relay this signal to Aft1 and Aft2. Interaction of Grx3/4 with Aft1 promotes dissociation of the transcriptional activator from its target DNA and export to the cytosol, leading to deactivation of Aft1/2-regulated genes. The exportin Msn5 facilitates iron-dependent export of both Aft1 and Aft2. We also observed Fe²⁺-dependent dimerization of Aft2 in vitro; however, the biological relevance still needs to be investigated in vivo (dotted lines).