Abstract
The cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm. CDF proteins are composed of two domains: the transmembrane domain, through which the cations are transported, and a regulatory cytoplasmic C-terminal domain (CTD). It was recently shown that the CTD of the CDF protein MamM from magnetotactic bacteria has a role in metal selectivity, as binding of different metal cations exhibits distinctive affinities and conformations. It is yet unclear whether the composition of the CTD binding sites can impact metal selectivity and if we can manipulate the CTD to response to other non-native metals in CDF proteins. Here we performed a mutational study of the model protein MamM CTD, where we exchanged the native metal binding residues with different metal-binding amino acids. Using X-ray crystallography and Trp-fluorescence spectrometry, we studied the impact of these mutations on the CTD conformation in the presence of non-native metals. Our results reveal that the incorporation of such mutations alters the domain response to metals in vitro, as mutant forms of the CTD bind metals differently in terms of the composition of the binding sites and the CTD conformation. Therefore, the results demonstrate the direct influence of the CTD binding site composition on CDF proteins structure and hence, function, and constitute a first step for rational design of MamM for transporting different metals in vivo.