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.
Highlights
The cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm
MamM C-terminal domain (CTD) was well characterized in vitro: its crystal structure revealed that it has the characteristic fold of CDF proteins; the CTD structure was shown to be crucial for the overall protein function; Divalent d-block metal cations (DDMCs) binding to the CTD causes a conformational change from a dynamic-apo form to a rigid, more closed V-shaped structure; and it binds different DDMCs d istinctively[14,16,21,22]
Comprehensive biophysical analysis of MamM CTD in the presence of Zn2+ and Fe2+ and in vivo studies showed that MamM CTD dimer binds three ions by two binding sites: a central binding site composed of D249 and H285 from both monomers, and two symmetrical peripheral binding sites, each composed of H264 from one monomer and E289 from the second monomer[14,22] (Figure S1)
Summary
The cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm. MamM CTD was well characterized in vitro: its crystal structure revealed that it has the characteristic fold of CDF proteins; the CTD structure was shown to be crucial for the overall protein function; DDMC binding to the CTD causes a conformational change from a dynamic-apo form to a rigid, more closed V-shaped structure; and it binds different DDMCs d istinctively[14,16,21,22]. Our results show that the CDFs CTD metal binding sites might be altered to change the response of this domain to different DDMCs in vitro, and to impact the regulation of the whole protein. This study enables a rational design of MamM mutants so as to change its metal selectivity and to control its regulation, and this would constitute a first step for a future synthesis of magnetic particles with different chemical properties in vivo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
