Cation diffusion facilitator (CDF) proteins are a conserved family of transmembrane transporters that ensure cellular homeostasis of divalent transition metal cations. Metal cations bind to CDF protein's cytoplasmic C‐terminal domain (CTD), leading to closure from its apo open V‐shaped dimer to a tighter packed structure, followed by a conformational change of the transmembrane domain, thus enabling transport of the metal cation. By implementing a comprehensive range of biochemical and biophysical methods, we studied the molecular mechanism of metal binding to the magnetotactic bacterial CDF protein MamM CTD. Our results reveal that the CTD is rather dynamic in its apo form, and that two dependent metal‐binding sites, a single central binding site and two symmetrical, peripheral sites, are available for metal binding. However, only cation binding to the peripheral sites leads to conformational changes that lock the protein in a compact state. Thus, this work reveals how metal binding is regulating the sequential uptakes of metal cations by MamM, and extends our understanding of the complex regulation mechanism of CDF proteins. Database Structural data are available in RCSB Protein Data Bank under the accession numbers: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G64, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G55, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G5E and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G6I (for CS, C267S, CS‐C267S and W247A, respectively).
Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain (TMD), in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal, or multiple metals, from the cytoplasm. Here, the model CDF protein MamM, from magnetotactic bacteria, was used to probe the role of the CTD in metal selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of; their binding sites, thermodynamics and binding-dependent conformation, both in crystal form and in solution. Furthermore, the results indicate that the CTD discriminates against Mn 2+ and provides the first direct evidence that CDF CTD's play a role in metal selectivity. KeywordsCation diffusion facilitator, magnetotactic bacteria, metal selectivity, protein-metal interactions, electron paramagnetic resonance (EPR) spectroscopy, pulsed electron double resonance (PELDOR) spectroscopy.
During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.