Characterisation of a cyanobacterial nickel sensor and a thermodynamic model of metal sensing

Abstract

The product of Synechocystis PCC 6803 open reading frame slr0176, InrS (Internal nickel responsive Sensor) a member of the CsoR-RcnR metalloregulatory family, has been characterised in the course of this work and found to regulate the expression of nrsD, encoding a nickel export protein, in a nickel dependent manner. InrS occludes a previously unidentified cryptic promoter in a nickel resistance operon. In addition to Ni(II) InrS is also competent to bind and respond to Zn(II), Co(II) Cu(II) and Cu(I) in vitro as determined by metal binding studies & fluorescence anisotropy. The factors that favour a response to nickel but disfavour responses to zinc and copper in vivo have been explored including determining allosteric coupling free energies of Ni(II) and Cu(I) binding. The cognate and non-cognate metal binding affinities of InrS, ZiaR (zinc-sensing SmtB family member), Zur (zinc-sensing Fur family member) and CoaR (cobalt-sensing MerR family) have been determined through competition with a range of metal chelators and infer that selective sensing of zinc and nickel can be dictated by the relative affinities of the metalloregulators but selective cobalt sensing cannot. The primary metal coordination sphere of InrS has been investigated through a combination of site directed mutagenesis and metal binding studies. Three of four Ni(II) ligands constituting the square planar Ni(II) site have been identified and mutant variants with weakened Ni(II) affinities produced. InrS displays metal binding properties characteristic of the copper sensing sub-branch of this protein family thought to be a consequence of the primary coordination sphere. Results of this work have been used to refine predictions as to the metal(s) sensed by uncharacterised members of this protein family based on the predicted secondary rather than primary coordination sphere. An alternative allosteric network involving a secondary coordination sphere hydrogen bond in nickel sensing CsoR-RcnRs is proposed.