Structural and spectroscopic insights into BolA-glutaredoxin complexes

Abstract

BolA proteins are defined as stress-responsive transcriptional regulators, but they also participate in iron metabolism. Although they can form [2Fe-2S]-containing complexes with monothiol glutaredoxins (Grx), structural details are lacking. Three Arabidopsis thaliana BolA structures were solved. They differ primarily by the size of a loop referred to as the variable [H/C] loop, which contains an important cysteine (BolA_C group) or histidine (BolA_H group) residue. From three-dimensional modeling and spectroscopic analyses of A. thaliana GrxS14-BolA1 holo-heterodimer (BolA_H), we provide evidence for the coordination of a Rieske-type [2Fe-2S] cluster. For BolA_C members, the cysteine could replace the histidine as a ligand. NMR interaction experiments using apoproteins indicate that a completely different heterodimer was formed involving the nucleic acid binding site of BolA and the C-terminal tail of Grx. The possible biological importance of these complexes is discussed considering the physiological functions previously assigned to BolA and to Grx-BolA or Grx-Grx complexes.

Keywords: BolA; Complexes; Glutaredoxin; Glutathione; Iron Metabolism; Iron-Sulfur Protein; Redox Regulation; Thiol.

FIGURE 1.

BolA overall fold. A, the Phe-119 and His-144 invariant residues and the β-turn are plotted on the x-ray structure of AtBolA1. The potential nucleic acid binding region containing the HTH structural motif is in yellow, and the [H/C] loop containing the putative [2Fe-2S] cluster ligands is in green. B, putative hydrogen bonds involving Arg-127 in AtBolA1.

FIGURE 2.

Interaction of AtBolA2 with AtGrxS14. A, the left panel displays the conserved face of BolAs, and the right panel displays the variable face of BolAs. Residue conservation ranges from 0% (cyan) to 100% (purple). RHR signature (blue circle) and putative ligands of the [2Fe-2S] cluster (green circle) are also highlighted on AtBolA2 surface. B, electrostatic potential of AtBolA2 generated by APBS (57) and showing negative charges in red and positive charges in blue. C, mapping of the residues of AtBolA2 (red) involved in the interaction with AtGrxS14, as shown by NMR. D, overlay of ¹H,¹⁵N HSQC spectrum of AtBolA2 (blue) and ¹H,¹⁵N HSQC spectrum of AtGrxS14-BolA2 at a 1:1 ratio (red). The assigned peaks are indicated with the residue number and one- letter code. E, zoom of the area delimited by dashed lines in D. The arrows indicate the direction in which the amide peak shifts upon the addition of AtGrxS14.

FIGURE 3.

Influence of AtBolA1 on AtGrxS14 intrinsic fluorescence. A, representative experiment showing the changes in AtGrxS14 fluorescence in the presence of increasing concentrations of AtBolA1. B, decrease of fluorescence emission at 337 nm was plotted against AtBolA1 concentration to determine the K_d value using a hyperbola equation. The data are represented as mean ± S.D. of three separate experiments. AU, arbitrary units.

FIGURE 4.

Glutaredoxin-BolA_H holo-heterodimer. A, model of the Grx-BolA holo-heterodimer with Rieske-type [2Fe-2S] coordination built using human Grx5 (PDB entry 2WUL) (58) and C. burnetii BolA, which coordinates a cobalt atom via His-29 and His-64 (PDB entry 3TR3). These residues are ideally positioned to coordinate an iron atom of a [2Fe-2S] cluster instead of cobalt, and they were used to orientate the BolA domain in the heterodimeric model. B, UV-visible absorption and CD spectra of AtGrxS14-BolA1 heterodimers. ϵ and Δϵ values are expressed per AtGrxS14-BolA1 heterodimer, and the arrow indicates the Soret band from a trace heme impurity. AU, arbitrary units.

FIGURE 5.

Spectroscopic studies of AtGrxS14-BolA1 holo-heterodimers. A, UV-visible absorption and CD spectra of oxidized (solid line) and dithionite-reduced (broken line) reconstituted AtGrxS14-BolA1 complex. ϵ and Δϵ values are expressed per AtGrxS14-BolA1 heterodimer, and the intense band at 314 nm in the reduced absorption spectrum arises from excess dithionite. The inset shows the X-band EPR spectrum of the dithionite-reduced sample recorded at 10 K and 9.581 GHz, with a microwave power of 5 milliwatts and a modulation amplitude of 0.65 millitesla. B, comparison of the CD spectra of the [2Fe-2S]²⁺ centers in the AtGrxS14-BolA1 and ScGrx3-Fra2 heterodimers and the AtGrxS14 homodimer. Δϵ values are expressed per [2Fe-2S] ²⁺ cluster. C, comparison of the resonance Raman spectra of the [2Fe-2S]²⁺ centers in the AtGrxS14-BolA1 and ScGrx3-Fra2 heterodimers and the AtGrxS14 homodimer. Spectra were recorded using 488-nm laser excitation. Samples were ∼2 mm in the [2Fe-2S] cluster and were in the form of a frozen droplet at 17–22 K. Each spectrum is the sum of 100 scans, with each scan involving photon counting for 1 s at 0.5 cm⁻¹ increments with 6 cm⁻¹ spectral resolution. Bands due to lattice modes of ice have been subtracted from all spectra. D, comparison of the X-band EPR spectra of the dithionite-reduced [2Fe-2S]¹⁺ centers in the AtGrxS14-BolA1 and ScGrx3-Fra2 heterodimers. EPR conditions: microwave frequency, 9.581 GHz; modulation amplitude, 0.65 millitesla; microwave power, 5 milliwatts; temperature, 10 K. Spin quantification of both EPR signals indicates 1.0 ± 0.1 spins per [2Fe-2S] cluster.

FIGURE 6.

Formation of BolA-Grx complexes. A, comparison of AtGrxS14-BolA apo- and holo-heterodimers. The [2Fe-2S]-bridged AtGrxS14-BolA1 holo-heterodimer model was built from the structures of individual proteins. The model of AtGrxS14-BolA2 apo-heterodimer was obtained by NMR-based docking from residues (colored in blue) presenting high chemical shift variations upon partner addition. Grxs are colored in green, BolA are in red, and glutathione is in yellow. B, recapitulative scheme showing the formation of BolA-BolA and Grx-BolA complexes. From the current literature, there are two pathways to form Grx-BolA holo-heterodimers. Grx-BolA apo-heterodimers can accept a [2Fe-2S] cluster from IscAs or possibly other Fe-S assembly components (pathway 1) (50). Alternatively BolA can displace a Grx monomer from holo-homodimeric Grxs as shown for the ScGrx3-Fra2 couple (pathway 2) (7). In principle, the reactions are reversible, and monomeric BolAs or Grx-BolA apo-heterodimers could be reformed by disruption (7) of the Grx-BolA holo-heterodimer Fe-S cluster or its transfer to an acceptor. Due to the presence of a cysteine residue, BolA_Cs can form disulfide bridges either as covalent dimers or as glutathionylated forms, both forms being reversibly reduced by Grxs (10). The physiological relevance of each form has not been always elucidated, but interconversion can affect all assumed functions of both proteins (DNA binding, iron-sensing, or Fe-S cluster maturation).