Copper (I) or (II) Replacement of the Structural Zinc Ion in the Prokaryotic Zinc Finger Ros Does Not Result in a Functional Domain

Abstract

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper’s interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.