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.