Evolution of metal(loid) binding sites in transcriptional regulators

Abstract

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys(15), Cys(16), and Cys(55). This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

FIGURE 1.

Multiple alignment of CgArsR homologues and homology modeling. A, representative ArsR homologues (accession numbers in parentheses) are from plasmid pI258 CadC (P20047), Synechocystis sp. PCC 7942 SmtB (P30340), plasmid R773 ArsR (P15905), C. glutamicum CgArsR1 (YP_225794) and CgArsR2 (NP_599514), A. ferrooxidans AfArsR (AAF69241), and Mycobacterium tuberculosis H37Rv CmtR (NP_216510). The structure-based multiple alignment was calculated using 3DCoffee (Pubmed ID 15201059). B, modeling of As(III)-free CgArsR was performed with SWISS-MODEL (Pubmed ID 12824332) (26), using the crystal structure of CadC (9) as template. The two monomers are colored in purple (subnunit a) and green (subunit b), respectively. The cysteines are shown in ball-and-stick models. The DNA binding domain is α4-turn-α5. The model was drawn using PyMOL (27). The side chain sulfur atoms of Cys¹⁵, Cys¹⁶, and Cys⁵⁵ are shown as yellow spheres. C, binding is proposed to occur in two steps: 1) As(III) (blue sphere) binds first to the thiolates of Cys⁵⁵ of one subunit and either Cys¹⁵ or Cys¹⁶ (shown here for only Cys¹⁵) in α1 of the other subunit, forming a low affinity S₂O site, and 2) the end of α1 unravels to allow the adjacent cysteine residue to become the third ligand to As(III), forming a high affinity S₃ site. The model of the As(III)-bound form was built by manually adjusting the N termini so that the geometry of As(III) coordination derived from the EXAFS results.

FIGURE 2.

EXAFS and Fourier transforms of CgArsR1 XAS data. EXAFS spectra of CgArsR1 with bound As(III) (A) and the corresponding Fourier transform (B) are shown as dashed lines, with the simulations of EXAFS and FT data shown as solid lines. A schematic representation of the putative coordination environment in CgArsR1 is shown in C. Sample preparation was as described under “Materials and Methods.”

FIGURE 3.

In vivo regulation of egfp2(gfp) expression from the arsB promoter using CgArsR1 and the seven Cys mutants in the presence or absence of As(III) or Sb(III). Construction of C. glutamicum 2Δars, further integration of arsR1 derivatives, mobilization of the reporter gene (gfp), sample preparation, and fluorescence measurements were performed as described under “Materials and Methods.” Strains C. glutamicum 2Δars (Control), 2Δars containing the arsR1 integrated (2Δarswt; WT), and the single (C15S, C16S, and C55S), double (C15S/C16S, C15S/C55S, and C16S/C55S), or triple (C15S/C16S/C55S) arsR1 mutant integrated were analyzed in the absence of arsenite (left bars) or the presence of 10 μM (middle bars) or 30 μm (right bars) As(III) (A) or in the absence (left bars) or presence of 10 μm (middle bars) or 30 μm (right bars) Sb(III) (B).

FIGURE 4.

Electrophoretic mobility shift assays of CgArsR-DNA binding. The igR1B1 PCR-amplified band (150 bp) was assayed in the presence (+) or absence (-) of CgArsR1 or CgArsR2 at the indicated concentrations of protein. In each case, a retarded band was observed upon PAGE using a 10% nondenaturating gel when the repressor protein was present.

FIGURE 5.

DNA footprinting. A, DNase I footprinting was performed as described under “Materials and Methods” using WellRED D3 dye-labeled double-stranded DNA. The sizes of major fragments in nucleotides are shown at the top of the peaks. The sequences bound to CgArsR1 are shown between the arrows. The positions -7, -37, -47, and -77 indicate nucleotides upstream of the start of the arsB gene. B, the sequence of the double-stranded DNA is shown with the binding sequences boxed. The highlighted sequences indicate a palindromic repeat in the intervening region between the arsR and arsB genes of the ars1 operon. The start sites of the arsR and arsB genes are indicated.

FIGURE 6.

CgArsR1 binding to DNA assayed by fluorescence anisotropy. DNA binding assays were performed as described under “Materials and Methods.” A, fluorescently labeled double-stranded DNA was titrated with the indicated concentrations of wild type CgArsR1 (•), methyl methanethiosulfonate-modified wild type CgArsR1 (▴), C15S (○), C16S (▾), C55S(▵), C15S/C16S (▪), C15S/C55S (□), C16S/C55S (⋄), C15S/C16S/C55S (♦), and wild type CgArsR1 preincubated with 0.3 mm sodium arsenite (▿). B, double-stranded DNA with bound wild type CgArsR1 was titrated with the indicated concentrations of sodium arsenite without GSH in the assay buffer (•) and with 2 mm GSH (○), C16S (▾), or methyl methanethiosulfonate-modified wild type CgArsR1 (▵) titrated with the indicated concentrations of sodium arsenite. C, double-stranded DNA with bound wild type CgArsR1 (•), CgArsR1 with 2 mm GSH (○), or C16S (▾) titrated with the indicated concentrations of potassium antimonyl tartrate.

FIGURE 7.

Reaction of CgArsR1 with dibromobimane. Wild type CgArsR1 (lanes 1 and 2) and single mutants C15S (lanes 3 and 4), C16S (lanes 5 and 6), and C55S (lane 7 and 8) were analyzed by SDS-PAGE on 16% polyacrylamide gels with (lanes 1, 3, 5, and 7) or without (lanes 2, 4, 6, and 8) reaction with dibromobimane. The gels were stained with Coomassie Blue (A) and visualized on a transilluminator for fluorescence (B). The positions of the 13-kDa monomer and 26-kDa dimer are indicated by the arrows.

FIGURE 8.

Location of metal(loid) binding sites in ArsR/SmtB repressors.Metal(loid) binding sites in members of the ArsR/SmtB family of repressor proteins are shown on a surface model of the CadC aporepressor structure either by coloring CadC residues corresponding to each binding site as identified from the structure-based alignment (Fig. 1A) or, in the case of CmtR, by overlaying the two structures. The S₃ As(III) binding site of the R773 ArsR (red) formed within each monomer overlaps with the corresponding S₄ Cd(II) binding site of CadC (yellow) formed between the N terminus of one subunit and the DNA binding domain of the other subunit. Zn(II) binding sites of CadC and SmtB (cyan) formed between the antiparallel C-terminal α6 helices also overlap. The S₃ binding sites of CgArsR1 (green), CmtR (blue), and AfArsR (purple) are at a variety of locations distributed over the surface of the repressor.