Convergent evolution of a new arsenic binding site in the ArsR/SmtB family of metalloregulators

Abstract

Acidithiobacillus ferrooxidans has an arsenic resistance operon that is controlled by an As(III)-responsive transcriptional repressor, AfArsR, a member of the ArsR/SmtB family of metalloregulators. AfArsR lacks the As(III) binding site of the ArsRs from plasmid R773 and Escherichia coli, which have a Cys(32)-Val-Cys(34)-Asp-Leu-Cys(37) sequence in the DNA binding site. In contrast, it has three cysteine residues, Cys(95), Cys(96), and Cys(102), that are not present in the R773 and E. coli ArsRs. The results of direct As(III) binding measurements and x-ray absorption spectroscopy show that these three cysteine residues form a 3-coordinate As(III) binding site. DNA binding studies indicate that binding of As(III) to these cysteine residues produces derepression. Homology modeling indicates that As(III) binding sites in AfArsR are located at the ends of antiparallel C-terminal helices in each monomer that form a dimerization domain. These results suggest that the As(III)-S(3) binding sites in AfArsR and R773 ArsR arose independently at spatially distinct locations in their three-dimensional structures.

FIGURE 1. Multiple alignment of AfArsR homologues

Representative ArsR homologues (accession numbers in parentheses) are from: A. ferrooxidans (AAF69241), Acidothiobacillus caldus (AAX35682), Comamonas testosteroni KF-1 (ZP_01519260), Methylobacillus flagellatus KT (ABE49761), Alcaligenes faecalis (AAS45114), Leptospirillum ferriphilum (AAY85166), Sinorhizobium meliloti 1021 (NP_385183), pI258 CadC (P20047), Synechocystis sp. PCC 6803 SmtB (BAA10706), Mycobacterium tuberculosis H37Rv CmtR (NP_216510), and plasmid R773 ArsR (P15905). Cysteine residues 95, 96, and 102 are indicated. The multiple alignment was calculated with CLUSTAL W (21).

FIGURE 2. Contribution of AfArsR cysteine residues to metalloregulation in vivo

Expression of a lacZ reporter gene was assayed as described under “Materials and Methods.” Cells of E. coli strain ACSH501^q bearing plasmids with wild type arsR, C95S, C96S, C102S, or the C95S/C96S double mutant in trans with reporter plasmid pACYC184lacZ-arsO were grown with 0.2% glucose (left bars), 0.2% arabinose (middle bars), or 0.2% arabinose and 25 μM sodium arsenite (right bars).

FIGURE 3. Determination of the molecular mass of AfArsR

Proteins were separated by gel filtration, as described under “Materials and Methods.” Molecular weight markers were ribonuclease, chymotrypsinogen A, ovalbumin, and albumin. The elution position of wild type and mutant AfArsRs is indicated by the arrow.

FIGURE 4. Circular dichroism spectroscopy of wild type and mutant AfArsRs

CD spectra were collected with wild type AfArsR (○), or wild type AfArsR with 30 μM sodium arsenite (●), C95S/C96S (▼), C96S (△), and C102S (□), respectively. The differences below 200 nm are likely due to slight differences in protein concentration.

FIGURE 5. Metalloid binding to AfArsR

Binding of (A) sodium arsenite or (B) potassium antimonyl tartrate was assayed as described under “Materials and Methods” to wild type AfArsR (○), MMTS-modified wild type AfArsR (●), C102S (△), MMTS-modified C102S (▲), C95S (□), C96S (▽), and C95S/C96S double mutant (■).

FIGURE 6. EXAFS and Fourier transforms of ArsR XAS data

EXAFS spectra of (A) wild type AfArsR and (D) C102S, both with bound As(III). The corresponding Fourier transforms (B and E) are shown as solid lines, with the simulations of EXAFS and Fourier transform data shown as dashed lines. Sample preparation was as described under “Materials and Methods.” A summary of the fitting results is shown in Table 1. C and F show models of the A_s-S₃ site of the wild type and the A_S-S₂O site of C102S, respectively.

FIGURE 7. AfArsR binding to DNA assayed by mobility shift assays

DNA binding assays were performed as described under “Materials and Methods.” Left, binding in the presence (+) or absence (−) of 0.1 mM sodium arsenite. Right, binding in the presence of varying concentrations (0, 100, 300, or 1000 mM) of sodium arsenite. Added proteins were none (control), wild type AfArsR, C95S, C96S, or C102S. Lanes M show a DNA ladder, with representative sizes indicated.

FIGURE 8. DNA footprinting

Top, DNase I footprinting was performed as described under “Materials and Methods” using light-sabre green fluorescently labeled double-stranded DNA. The sizes of major DNA fragments in nucleotides are shown at the top of the peaks. The sequence protected by AfArsR is shown between the arrows, with the positions of −60, −73, and −86 nucleotides relative to the start of the arsB gene indicated. Bottom, the sequence of the double-stranded DNA is shown, with the region protected by AfArsR boxed. The start sites for the arsR and arsB genes are indicated, with the −10, −60, −73, and −86 nucleotide positions relative to the start of arsB indicated.

FIGURE 9. AfArsR 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 AfArsR (●), wild type AfArsR plus a 6-fold excess of sodium arsenite (○), C102S (▲), C102S plus a 6-fold excess of sodium arsenite (▽), C95S (■), C95S plus a 6-fold excess of sodium arsenite (□). B, double-stranded DNA with bound wild type AfArsR was titrated with the indicated concentrations of potassium antimonyl tartrate (○), sodium arsenite (▽), or cadmium chloride (□). C, fluorescently labeled double-stranded DNA was titrated with the indicated concentrations of wild type AfArsR (●), MMTS-modified wild type AfArsR (○), C102S (▼), and MMTS-modified C102S (△). D, double-stranded DNA with bound wild type AfArsR (●), MMTS-modified wild type AfArsR (△), C102S (▼), or MMTS-modified C102S (○) was titrated with the indicated concentrations of sodium arsenite.

FIGURE 10. As(III) binding model of AfArsR

Modeling of As(III)-free AfArsR was performed with the Modeler 8v1 auto mode (22), using the crystal structure of CadC (6) as template. The model of the As(III)-bound form was built by manually adjusting the C termini so that the geometry of As(III) coordination from Fig. 6 was satisfied. The two monomers are colored red and green, respectively. The cysteines are shown in sticks. The As(III)s are shown as blue spheres. The model was drawn using PyMOL (23). A, the aporepressor binds two arsenic atoms at the ends of the α5 helix. B, binding is proposed to occur in three steps: 1) As(III) binds first to the thiolate of either Cys⁹⁵ or Cys⁹⁶ in the α5 helix; 2) the end of the helix unravels to allow the adjacent cysteine residue to become a second ligand to As(III), forming a low affinity S₂O site; and 3) Cys¹⁰² in the flexible C terminus forms the third ligand to the As(III), forming a high affinity S₃ site.