Assessing the functional selectivity of an arsenic sensing protein in vitro and in vivo

Abstract

The homodimeric bacterial ArsR proteins respond to As^III and Sb^III. Binding of the metalloids at coordination sites formed by three cyteine residues triggers an allosteric mechanism, leading to the release of the repressor ArsR protein from the operator DNA. Our study is focussed on the functional selectivity of ArsR from Acidithiobacillus ferrooxidans (AfArsR) both in vitro and in vivo. Binding of the inducers As^III and Sb^III, as well as non-cognate metal ions Hg^II, Pb^II, Cd^II and Zn^II to AfArsR were characterized by UV absorption titrations, ^199mHg Perturbed Angular Correlation (PAC) of γ-rays spectroscopy, and Electrospray Ionization Mass Spectrometry (ESI-MS). The data indicate that metalloid binding at the two metalation sites is sequential. Correspondingly, Electrophoretic Mobility Shift Assays (EMSA) demonstrated that ca. 1.0 equivalent of As^III per protein dimer leads to a significant dissociation of the protein-DNA complex, suggesting that activation of the protein dimer requires the binding of only one As^III. Contrary to this, an Sb^III:AfArsR dimer concentration ratio higher than 1 was required to induce dissociation of the DNA from the DNA-protein complex. The divalent thiophilic metal ions bind strongly to the protein, but do not induce dissociation of the DNA-protein complex. This demonstrates that the inherent, molecular-level metalloid selectivity of AfArsR is not achieved via binding affinity differences. Interestingly, when combined with the inducer AsIII, HgII was able to prevent the dissociation of the AfArsR dimer-DNA complex, however, this inhibiting effect of Hg^II was reversed by the addition of dimercaptosuccinic acid (DMSA). Selective As^III/Sb^III response of AfArsR was also observed in vivo in a bioreporter construct. The divalent metal ions, even Hg^II, did not compromise this effect, presumably due to the presence of competing thiol-containing Hg^II-binders in the cell, in analogy to the presence of DMSA in vitro. Experiments on mutant proteins confirmed that Cys95 and Cys96 are essential for and that C102 affects the protein function. Moreover, our data indicate that the His97Asp mutation also affects the metalloid response of the protein in vivo.