The Conserved CDC Motif in the Yeast Iron Regulator Aft2 Mediates Iron-Sulfur Cluster Exchange and Protein-Protein Interactions with Grx3 and Bol2

Abstract

The Saccharomyces cerevisiae transcriptional activator Aft1 and its paralog Aft2 respond to iron deficiency by upregulating expression of proteins required for iron uptake at the plasma membrane, vacuolar iron transport, and mitochondrial iron metabolism, with the net result of mobilizing iron from extracellular sources and intracellular stores. Conversely, when iron levels are sufficient, Aft1 and Aft2 interact with the cytosolic glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, which promote Aft1/2 dissociation from DNA and subsequent export from the nucleus. Previous studies unveiled the molecular mechanism for iron-dependent inhibition of Aft1/2 activity, demonstrating that the [2Fe-2S]-bridged Grx3-Bol2 heterodimer transfers a cluster to Aft2, driving Aft2 dimerization and dissociation from DNA. Here we provide further insight into the regulation mechanism by investigating the roles of conserved cysteines in Aft2 in iron-sulfur cluster binding and interaction with [2Fe-2S]-Grx3-Bol2. Using size exclusion chromatography and circular dichroism spectroscopy, these studies reveal that both cysteines in the conserved Aft2 Cys-Asp-Cys motif are essential for Aft2 dimerization via [2Fe-2S] cluster binding, while only one cysteine is required for interaction with the [2Fe-2S]-Grx3-Bol2 complex. Taken together, these results provide novel insight into the molecular details of iron-sulfur cluster transfer from Grx3-Bol2 to Aft2 which likely occurs through a ligand exchange mechanism. Loss of either cysteine in the Aft2 iron-sulfur binding site may disrupt this ligand exchange process leading to isolation of a trapped Aft2-Grx3-Bol2 intermediate, while replacement of both cysteines abrogates both the iron-sulfur cluster exchange and the protein-protein interactions between Aft2 and Grx3-Bol2.

Introduction

In the yeast Saccharomyces cerevisiae, the transcriptional activators Aft1 and Aft2 control the expression of iron uptake and storage genes and help remodel metabolic pathways in response to iron starvation [1–4]. Aft1 and Aft2 are paralogs that have both overlapping and independent functions and bind to similar promoter sequences [5, 6]. When iron levels are low, Aft1 and Aft2 accumulate in the nucleus and activate the iron regulon; whereas Aft1 and Aft2 are both exported to the cytosol during iron sufficiency, causing deactivation of the iron regulon [7]. Genetics studies have revealed that iron-dependent inhibition of Aft1/Aft2 activity is linked to mitochondrial iron-sulfur (Fe-S) cluster biogenesis and a signaling pathway that includes the CGFS monothiol glutaredoxins Grx3 and Grx4 and the BolA-like protein Bol2 (previously known as Fra2) [8, 9]. Inhibition of Aft1/2 by the Grx3/4-Bol2 signaling pathway is dependent on a conserved, iron-responsive Cys-Asp-Cys motif located approximately 50 amino acids downstream of the zinc-finger DNA binding domains in Aft1 and Aft2. In the absence of these signaling factors or upon substitution of either Cys in the CDC motif, Aft1 and Aft2 are constitutively active, stimulating intracellular iron overload and oxidant sensitivity [3, 10–13].

Recent spectroscopic and biochemical analyses have unveiled the molecular mechanism for inhibition of Aft1/Aft2 activity in response to iron. Two Aft2 monomers were shown to form a [2Fe-2S]-bridged homodimer that has reduced DNA binding affinity compared to the apo, monomeric transcription factor [14]. In this regulation model, the [2Fe-2S] cluster is bound at the conserved CDC motif in Aft2 and delivered to Aft2 via a [2Fe-2S]-bridged heterodimer formed by Grx3 (or Grx4) and Bol2 [15–17]. Grx3 and Grx4 also form [2Fe-2S]-bridged homodimers in vivo and in vitro [12, 18]; however, the [2Fe-2S]-Grx3 homodimer was shown to be inefficient in delivery of the Fe-S cluster to Aft2, highlighting the essential role of Bol2 in Aft2 recognition and cluster transfer [14].

To better understand the mechanism for [2Fe-2S] transfer from Grx3-Bol2 to Aft2, we generated Aft2 variants with Ala substitutions in one or both Cys in the iron-responsive CDC motif and examined their interactions with [2Fe-2S]-Grx3-Bol2 using size exclusion chromatography, circular dichroism, and UV-visible absorption spectroscopy. Comparison of the spectroscopic changes upon titration of [2Fe-2S]-Grx3-Bol2 with these Aft2 variants provides evidence for the requirement for both Cys to form a [2Fe-2S]-bridged Aft2 homodimer with a coordination environment that is distinct from [2Fe-2S]-Grx3-Bol2. However, only one Cys is required to mediate protein-protein interactions between Aft2 and Grx3-Bol2 allowing formation of an Aft2-Grx3-Bol2 heterocomplex. These studies therefore provide new insight into the molecular details that govern regulation of iron metabolism in this model eukaryote.

Materials and Methods

Plasmid Construction

Construction of pET30a-Aft2(1–204) (WT or C187A) was described previously [14]. Aft2(1–204) is a truncated form of the transcription factor that includes the DNA binding zinc-finger domain and the conserved iron-responsive CDC motif. Aft2(1–204) variants, C189A and C187A/C189A, were created by site-directed mutagenesis of pET30a-Aft2(1–204) using primers listed in Table 1.

Table 1.

Primers used in this study. Codons altered by site-directed mutagenesis are shown in bold.

Primer Name	Primer Sequence
C189A Forward	5′-CCAATTATTTCTTGTGACGCTGGGTTAACC-3′
C189A Reverse	5′-GGTTAACCCAGCGTCACAAGAAATAATTGG-3′
C187A/C189A Forward	5′-CCAATTATTTCTGCTGACGCTGGGTTAACC-3′
C187A/C189A Reverse	5′-GGTTAACCCAGCGTCAGCAGAAATAATTGG-3′

Protein Expression and Purification

Grx3, Bol2, and [2Fe-2S]-Grx3-Bol2 were prepared as previously described [15, 16]. E. coli BL21(DE3) cells transformed with Aft2(1–204) (hereby referred to as Aft2) expression plasmids were grown at 37 °C in 2 L LB with 30 μg/ml kanamycin. The cultures were induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) at OD₆₀₀ 0.6 – 0.8 and grown at 20 °C for 18 h. Cells were collected by centrifugation and resuspended in 50 ml of Buffer A [50 mM Tris-HCl, pH 7.4, 300 mM NaCl], with 5 mM Tris[2-carboxyethyl] phosphine (TCEP) and 1 mM phenylmethanesulfonyl fluoride (PMSF) added prior to purification. The cells were broken by intermittent sonication and the cell debris was removed by centrifugation. The cell-free extract was loaded onto a 20-ml HiPrep 16/10 heparin FF column (GE Healthcare) equilibrated with Buffer A with 5 mM TCEP. The protein was eluted with a NaCl gradient and the purest fractions containing Aft2, as judged by SDS-PAGE, were pooled and concentrated to 1 ml. The concentrated sample was further loaded onto a HiLoad Superdex 75 gel filtration column (GE Healthcare) equilibrated with Buffer A containing 5 mM TCEP. Aft2 fractions were collected and concentrated to ~250 μl with the addition of 5% glycerol and stored at −80 °C. Prior to interaction studies with cluster-containing proteins, TCEP was removed from purified Aft2 inside a glovebox (Coy Laboratory Products, Inc.) by buffer exchange using a 5-ml HiTrap desalting column (GE Healthcare) equilibrated with Buffer A.

Analytical and Spectroscopic Methods

Protein concentrations were determined by the Bradford assay (Bio-Rad) using bovine serum albumin as the standard. Iron and acid-labile sulfur concentrations were measured as previously described [16]. Size exclusion chromatography (SEC) analyses were performed on a Superdex 200 10/300 GL column equilibrated with Buffer B and calibrated with a gel filtration calibration kit (GE Healthcare). Chromatograms were obtained by monitoring absorbance at 280 nm. The same set of fractions was collected for all proteins subjected to SEC and was analyzed by SDS-PAGE in order to compare relative elution times. Circular dichroism (CD) and UV-visible absorption spectra were recorded under anaerobic conditions using a Jasco J-815 spectropolarimeter or a Beckman DU-800 spectrophotometer, respectively.

CD Monitored Titration of [2Fe-2S] Grx3-Bol2 with Aft2

The titration of [2Fe-2S] Grx3-Bol2 with Aft2 was monitored under anaerobic conditions at room temperature using UV-visible CD spectroscopy. The [2Fe-2S] Grx3-Bol2 sample used in the titrations typically contained 0.9–1.05 [2Fe-2S] cluster per heterodimer as determined by Bradford, iron, and acid labile sulfide analysis. Reactions were carried out in Buffer B [50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM GSH], with the [2Fe-2S]²⁺ cluster concentration kept constant at 40 μM and [Aft2] varied from 10 to 340 μM. After addition of Aft2, samples were anaerobically equilibrated for 15 min at room temperature prior to recording CD spectra.

Size Exclusion Chromatography Analysis of [2Fe-2S]-Grx3-Bol2 and Aft2

To examine the protein-protein interactions between [2Fe-2S] Grx3-Bol2 heterodimer and Aft2 variants (WT, C187A, C189A, and C187A/C189A), the protein samples were mixed together at a 2:1 ratio of Aft2 to [2Fe-2S] cluster with the cluster concentration at 40 μM in 500 μl. All the samples were prepared in Buffer B under anaerobic conditions (O₂ < 5 ppm) in the glovebox and incubated at room temperature for 15 min prior to size exclusion chromatography. Reducing SDS-PAGE analysis was performed on collected fractions.

Isolation of Aft2 after Interaction with [2Fe-2S] Grx3-Bol2

A 300-μl reaction mixture was prepared in Buffer B containing 400 μM apo-Aft2 (desalted prior to use) and 200 μM [2Fe-2S]²⁺-Grx3-Bol2 heterodimeric complex. After a 15-min incubation at room temperature, the reaction mixture was desalted into Buffer A with a 5-ml HiTrap desalting column and loaded onto a 1-ml HiTrap heparin HP column (GE Healthcare) equilibrated with Buffer A, followed by washing. Aft2 was then eluted with Buffer C [50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM GSH] in one step. The fractions from the flow-through and eluate were pooled and concentrated to 250 – 350 μl for further analysis by biochemical, analytical, and spectroscopic methods.

Results and Discussion

Both Cys in the Aft2 CDC motif are required to significantly impact coordination of the [2Fe–2S] cluster in Grx3–Bol2

Our previous study provided evidence that Aft2 receives a [2Fe-2S] cluster from a [2Fe-2S]-bridged Grx3-Bol2 heterodimer to form a [2Fe-2S]-bridged Aft2 homodimer, presumably ligated by the conserved CDC motif in Aft2 [14]. To confirm and probe the individual roles of each Cys in binding and receiving the [2Fe-2S] cluster from Grx3-Bol2, we used site-directed mutagenesis to create single and double Cys to Ala mutations in the CDC motif and assessed the impact on the coordination environment of the [2Fe-2S] cluster using UV-visible CD spectroscopy. This optical technique is acutely sensitive to small differences in the [2Fe-2S] coordination environment, providing a convenient handle to compare and contrast effects of the Aft2 variants on the Fe-S cluster. As previously shown, titration of [2Fe-2S] Grx3-Bol2 with increasing Aft2 causes significant changes in the visible CD spectrum between 350 and 650 nm that are indicative of alterations in the cluster chirality and/or ligation environment (Fig. 1a). These changes are plotted as a function of Aft2 concentration in Figure 1d. In contrast, addition of the Aft2(C187A) variant to [2Fe-2S] Grx3-Bol2 had little effect on the CD spectrum, suggesting minimal perturbation in the cluster coordination (Fig. 1b and 1d). A similar outcome was noted for addition of Aft2(C187,189A) to [2Fe-2S] Grx3-Bol2 (data not shown). Interestingly, the Aft2(C189A) variant elicited some CD spectrum changes that were localized in the 340–470 nm region, suggesting a slight perturbation in the cluster coordination sphere (Fig. 1c). Thus, Cys187 may be binding at or near the cluster in the Aft2(C189A)-Grx3-Bol2 interaction.

Figure 1.

Titration of [2Fe-2S] Grx3-Bol2 with apo-Aft2 monitored by UV-visible CD spectroscopy. CD spectra for [2Fe-2S]²⁺ cluster-bound Grx3-Bol2 heterodimers (black line) titrated with 0.25–5-fold excess Aft2(WT) (a), Aft2(C187A) (b), or Aft2(C189A) (c) (gray lines). Colored lines in each graph (blue, pink, purple) correspond to 5-fold excess Aft2 added. Δɛ values are based on the [2Fe-2S]²⁺ cluster concentration (40 μM). d Difference in CD intensity between 400 and 473 nm (arrows in a) as a function of [Aft2] from CD spectra shown in (a-c).

A single Cys in the Aft2 CDC motif facilitates ternary complex formation with [2Fe–2S]–Grx3–Bol2

We next addressed the role of the Aft2 CDC motif in facilitating the protein-protein interactions between Aft2 and [2Fe-2S] Grx3-Bol2 using size exclusion chromatography. Aft2(WT) and the single and double Cys➔ Ala variants all elute as monomers at approximately 42 kDa, while [2Fe-2S]-Grx3-Bol2 alone elutes as a heterodimer at approximately 53 kD (Fig. 2, top) [14]. However, upon mixing Aft2(WT) with [2Fe-2S]-Grx3-Bol2 in a 2:1 ratio, the main peak in the chromatogram is shifted to a higher molecular mass (85 kD) corresponding to a [2Fe-2S]-bridged Aft2 dimer (Fig. 2, bottom) [14]. A portion of Grx3-Bol2 also elutes at a higher molecular mass with Aft2 suggesting formation of an Aft2-Grx3-Bol2 heterocomplex (Fig. S1a). Substitution of either Cys in the Aft2 CDC motif (C187 or C189) significantly reduced formation of the higher molecular mass peak at 85 kD (Fig. 2, bottom), confirming that both Cys residues are required for efficient dimer formation. However, SDS-PAGE analysis of the fractions collected from the SEC experiment suggest that both the C187A and C189A Aft2 variants still bind and interact with a portion of Grx3-Bol2 (Fig. S1b–c). In contrast, replacement of both Cys residues in the Aft2 CDC motif (C187A/C189A) caused nearly complete loss of interaction between Aft2 and Grx3-Bol2 (Fig. 2, bottom and Fig. S1d).

Figure 2.

Top, Size exclusion chromatograms of Aft2(WT), Aft2 Cys variants, and [2Fe-2S] Grx3-Bol2 run separately. Bottom, Size exclusion chromatograms of Aft2(WT) or Aft2 Cys variants mixed with [2Fe-2S] Grx3-Bol2 in a 2:1 ratio prior to loading. SDS-PAGE analysis of fractions collected from each chromatography run are shown in Supplementary Fig. 1.

To more easily characterize the complex formed between the Aft2 variants and [2Fe-2S]-Grx3-Bol2, we used heparin chromatography to separate Aft2 from Grx3-Bol2 since Aft2 binds to this column (Fig. 3, lanes 1–2), while [2Fe-2S]-Grx3-Bol2 does not (Fig. 3, lanes 3–4) [14]. SDS-PAGE analysis of the flow-through and eluate fractions for the Aft2 + [2Fe-2S]-Grx3-Bol2 mixtures confirms that a portion of Grx3 and Bol2 maintains an interaction with Aft2(WT) (lanes 5–6), Aft2(C187A) (lanes 7–8) and Aft2 (C189A) (lanes 9–10), presumably forming ternary complexes. However, this interaction is largely abrogated when both Cys are replaced in the Aft2(C187,189A) variant (lanes 11–12), similar to the result obtained in the SEC experiment (Fig. S1d).

Figure 3.

SDS-PAGE analysis following heparin chromatography separation of Aft2(WT) or [2Fe-2S]-Grx3-Bol2 alone, or Aft2(WT) or CDC variants mixed in a 2:1 ratio with [2Fe-2S]-Grx3-Bol2. The % Fe measured in the flow-through (F-T) vs. the eluate (E) for each mixture is shown below each lane. MW std, protein molecular weight standards; Fe-S, [2Fe-2S]-Grx3-Bol2. Aft2 = 23.3 kDa, Grx3 = 28.1 kDa, Bol2 = 14.0 kDa. Bol2 typically runs as two bands on the gel due to partial proteolysis [14,16].

We also measured the relative iron levels in the flow-through and eluate fractions for each Aft2 variant as a means to monitor cluster transfer between [2Fe-2S]-Grx3-Bol2 and Aft2. As noted previously [14], the iron content of the Aft2(WT)-containing eluate increases from 0 to 70% following interaction with [2Fe-2S]-Grx3-Bol2 with a parallel decrease in the iron content of the flow-through. However, the Aft2(C187A) and Aft2(C189A) variant fractions retain only 32% and 39%, respectively, of the total iron, even though the amounts of Grx3 and Bol2 proteins that coelute with each variant are similar to WT (Fig. 3, compare lanes 6, 8 and 10). The iron level in the eluate drops even further to 5% when both Cys are substituted, demonstrating that these Cys are critical for both iron binding and interaction with Grx3-Bol2.

Both Cys in the Aft2 CDC motif are required to complete Fe-S cluster transfer from [2Fe–2S]–Grx3–Bol2 to Aft2

To better gauge the oligomeric state of Aft2 and Grx3-Bol2 in the heparin column eluates following their interaction, these fractions were subjected to SEC analysis. The results shown in Figure 4a suggest that Aft2(WT) elutes primarily as a dimer and a higher molecular weight Aft2-Grx3-Bol2 heterocomplex. Monomeric Aft2 is also present but constitutes a smaller fraction of the total Aft2 protein. In contrast, the oligomeric state of each of the Aft2 single Cys variants is shifted primarily towards the monomeric form, consistent with the apo form of the protein. The lower intensity peak at the higher molecular mass (approximately 90–110 kDa) elutes as a complex somewhat larger that the Aft2 dimer, suggesting formation of an Aft2-Grx3-Bol2 ternary heterocomplex, which is confirmed by the SDS-PAGE analysis (Fig. 4a). Finally, the heparin eluate of the Aft2(C187,189A) + [2Fe-2S]-Grx3-Bol2 mixture is almost exclusively Aft2 monomers as expected in the absence of [2Fe-2S] cluster binding.

Figure 4.

(a) Top, Size exclusion chromatograms of eluates after heparin column separation for 2:1 mixtures of Aft2 variants + [2Fe-2S]-Grx3-Bol2. Bottom, SDS-PAGE analysis of the fractions collected for each mixture. (b) UV-visible absorption (top) and CD (bottom) spectra of heparin column eluates for 2:1 mixtures of Aft2 variants + [2Fe-2S] Grx3-Bol2 (colored solid lines), as compared to [2Fe-2S]-Grx3-Bol2 alone (black dashed line). Black, vertical dotted lines correspond to major UV-visible absorption peaks in [2Fe-2S]-Grx3-Bol2. The red vertical dotted lines and red arrows indicate the shift in these peaks in the Aft2(WT) sample (blue spectrum). ɛ and Δɛ values are normalized to protein concentrations.

Using CD and UV-visible absorption spectroscopy, we next compared the [2Fe-2S] cluster coordination environments in the heparin column eluates for each of the Aft2 variants following interaction with [2Fe-2S]-Grx3-Bol2. As shown in Figure 4b, the UV-visible and CD spectra of Aft2(WT) are clearly different from [2Fe-2S]-Grx3-Bol2. In the UV-visible absorption spectrum, the Grx3-Bol2 heterodimer has three distinct peaks at 320 nm, 392 nm and 435 nm that are attributed to S ➔ Fe(III) charge transfer transitions. These latter two peaks are red-shifted by 15–17 nm in the [2Fe-2S]-Aft(WT) complex to 409 nm and 450 nm (Fig. 4b, top). These differences are also manifested in significant changes in the wavelengths and magnitudes of the visible CD bands (Fig. 4b, bottom), suggesting a [2Fe-2S] cluster environment that is distinct from [2Fe-2S]-Grx3-Bol2. For each of the Aft2 single Cys variants, the UV-visible peaks are less intense, reflecting a lower [2Fe-2S] level, and the peak positions match the locations of the [2Fe-2S]-Grx3-Bol2 spectrum (Fig. 4b, top). A similar pattern is observed in the CD spectra for these variants, which resemble the spectra for the Aft2 + [2Fe-2S]-Grx3-Bol2 mixtures prior to separation by heparin chromatography (Fig. 1b–c). Taken together, these spectra suggest that Aft2(C187A) and Aft2(C189A) do not significantly perturb the Fe-S coordination environment in [2Fe-2S]-Grx3-Bol2 even though they form stable ternary complexes with the Fe-S cluster-bridged heterodimer. As expected, the double Cys➔Ala Aft2 variant has very weak UV-visible and CD signals, reflecting the low Fe content and lack of stable interaction with [2Fe-2S] Grx3-Bol2.

In conclusion, the biochemical and spectroscopic results presented herein support previous in vivo results confirming that the Aft1/2 CDC motif is key to the inhibition mechanism of this transcription factor, while revealing the molecular details of the protein-protein and metal-protein interactions that mediate this iron signaling pathway. Substitution of both Cys in the Aft2 CDC motif disrupts the in vivo interaction between Grx3/4 and Aft1/2 [13, 19], which is consistent with our in vitro protein-protein interaction analysis using chromatographic separation of the purified proteins. In addition, substitution of one or both Cys in the CDC motif prevents Aft1/2 oligomerization in vivo [20], which is supported by our spectroscopic and analytical analysis of the single and double Cys variants that do not form [2Fe-2S]-bridged Aft2 homodimers. Interestingly, we observe an Aft2-Grx3-Bol2 heterocomplex with the Aft2 single Cys variants which may represent a trapped Fe-S cluster transfer intermediate with a cluster coordination environment that is more similar to [2Fe-2S]-Grx3-Bol2 than [2Fe-2S]-Aft2(WT). This ternary complex is also observed in the interaction between Aft2(WT) and [2Fe-2S]-Grx3-Bol2, and thus may represent an intermediate in the Fe-S cluster transfer pathway. Taken together, these results suggest that Fe-S cluster transfer between Grx3-Bol2 and Aft2 (and presumably Aft1) involves a ligand exchange mechanism in which Cys187 in Aft2 may initiate the binding event, while Cys189 is required to complete the transfer and form the [2Fe-2S]-bridged Aft2 homodimer (Figure 5). Furthermore, the transfer is mediated via specific protein-protein interactions between Grx3-Bol2 and Aft2 that require at least one Cys in Aft2.

Figure 5.

Model for Fe-S cluster transfer between [2Fe-2S] Grx3-Bol2 and Aft2. Bol2 facilitates the interaction with Aft2 allowing access to the [2Fe-2S] cluster. Cys187 in Aft2 (in red) may bind at or near the cluster in the ternary [2Fe-2S] cluster-bound intermediate complex, displacing a ligand from Grx3, Bol2 or GSH (2 of these possible conformations are shown in the model). Cys189 in Aft2 and a second Aft2 molecule is required to complete the [2Fe-2S] transfer and form the [2Fe-2S]-bridged Aft2 homodimer.

Abbreviations

CD

Circular dichroism

CDC

Cys-Asp-Cys

Cys

Cysteine

Fe-S

Iron-sulfur

Grx

Glutaredoxin

GSH

Glutathione

IPTG

Isopropyl β-D-thiogalactoside

LB

Luria-Bertani medium

PMSF

Phenylmethanesulfonyl fluoride

SEC

Size exclusion chromatography

TCEP

Tris[2-carboxyethyl] phosphine

WT

Wild type