ZraP, the most prominent zinc protein under zinc stress conditions has no direct role in in-vivo zinc tolerance in Escherichia coli

Escherichia coli ZraP (zinc resistance associated protein) is the major Zn containing soluble protein under Zn stress conditions. ZraP is the accessory protein of a bacterial two-component, Zn2+ sensitive signal transduction system ZraSR. ZraP has also been reported to act as a Zn2+ dependent molecular chaperone. An explanation why ZraP is the major Zn protein under the stress condition of Zn2+ overload (0.2 mM) has remained elusive. We have recombinantly produced E. coli ZraP and measured Zn2+ and Cu2+ affinity in-vitro using Isothermal Titration Calorimetry. ZraP has a significantly higher affinity for Cu2+ than for Zn2+. Mutation of the conserved Cys102 to Ala or Ser resulted in a change of the oligomeric state of the protein. Mutation of the conserved His107 to Ala did not affect the zinc binding affinity or the oligomeric state of the protein. Deletion of the ZraP coding gene from the E. coli genome resulted in a phenotype with tolerance to very high zinc concentrations (up to 2.5 mM) that were lethal to wild type E. coli. These results exclude a direct role for ZraP in Zn2+ tolerance in E. coli. Escherichia coli Zrap (zinc resistance associated protein) is the major zinc protein under zinc stress. Metal affinity to Zrap was measured using Isothermal Titration Calorimetry. zraP disruption affects the bacterial growth. [Display omitted] •ZraP is the zinc resistance associated protein.•ZraP has medium affinity for Zn2+ and high affinity for Cu2+.•ZraP gene disruption results in more tolerance to millimolar (toxic) Zn2+ levels.•Mutation of the conserved Cys102 to Ala or Ser changed the oligomeric state.•Mutation of conserved His107 to Ala did not affect Zn2+ affinity or oligomeric state.