Models designed to study protein/metal ion interaction are helpful to provide insights into the rules of metal ion selectivity. In this context, the prokaryotic zinc finger family Ros/MucR offers an example of several naturally occurring homologues binding a structural zinc ion with coordination spheres characterized by different amino acid arrays. In particular, Ros87, the zinc binding domain of the protein Ros from A. tumefaciens, binds Zn(II) with a classical Cys2His2 coordination sphere, but most of its homologues show a substitution of the second cysteine by an aspartate. In this study, the binding properties to Zn(II), Co(II) and Cd(II) of the protein Ros87-C27D, Ros87 mutant with a CysAspHis2 coordination sphere, are investigated by means of UV-vis, CD and NMR spectroscopies. Dissociation constants, structural effects and the resulting mechanisms of folding are compared with the wild-type protein bearing the classical Cys2His2 coordination sphere. CysAspHis2 coordination sphere induces a two-state mechanism of folding in the presence of all three different metals, while, differently in the case of Ros87 complexed to Zn(II) or Co(II), the presence of the second cysteine in the coordination sphere leads to the formation of a stable metal binding folding intermediate. Our study underlines how the interplay between the different metal ions and the coordinating amino acid sets is determinant in defining the different Kds and the folding pathway of a given protein.
Dragone et al. (Thu,) studied this question.