Structure and Function of Bacterial CNNM Magnesium Transporters

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Magnesium (Mg2+) is the most abundant divalent cation inside cells and essential for a wide variety of biochemical activities. Although there is not a large gradient of Mg2+ across the cell membrane, increasing evidence suggests that changes in intracellular Mg2+ modulate cell growth through the requirement of many enzymes and metabolic sensors for Mg2+. The importance of cellular Mg2+ homeostasis is manifest in the large number of Mg2+ transporters both in bacteria and eukaryotes. In humans, hereditary diseases of magnesium uptake or deposition arise from mutations in transient receptor potential melastatin type 6 (TRPM6), Mg2+ transporter 1 (MagT1), solute carrier family 41 members (Slc41a1-3), claudin 16, and CNNMs. In bacteria, crystal structures for the Mg2+ transporters MgtE and CorA have revealed the mechanisms for ion selectivity and gating. The CNNM proteins are of particular interest as they are conserved across both eukaryotes and prokaryotes and play essential roles in Mg2+ homeostasis. The CNNM family is defined by a conserved transmembrane domain (DUF21 -- domain of unknown function 21) and a cytosolic cystathionine beta--synthase (CBS) pair domain. While associated with Mg2+ transport, it is unknown if CNNM proteins are themselves Mg2+ transporters or if they regulate transport by other proteins. In humans, mutations in CNNM proteins cause a variety of diseases related to Mg2+ and Ca2+ uptake or deposition. In lower eukaryotes and bacteria, mutations in CNNM proteins lead to resistance to metal toxicity and defects in Mg2+ transport. Our group has determined multiple structures of CNNM cytosolic domains and shown that they undergo a large conformational change upon Mg2+·ATP binding. Here, we propose to determine the structural basis of bacterial CNNM protein function and regulation. We have two aims: 1. Structural studies. We have cloned and expressed DUF21--containing proteins from over 20 species. Several proteins are well expressed and crystallize under multiple conditions. We have also used negative stain electron microscopy (EM) to study one bacterial protein. We will determine the structure of a CNNM bacterial ortholog by either EM or X--ray crystallography. 2. Functional studies. We will characterize the function and conformational changes of CNNM proteins using biophysical methods and functional assays. These studies will address the question of whether DUF21 domains directly or indirectly mediate ion transport and reveal how their cytosolic domains regulate their function. The DUF21 domain of CNNM proteins is the largest family of domains of unknown function and there are no known structures. There are well over 50,000 CNNM orthologs known in plants, animals and bacteria yet we do not understand their precise biochemical function. The work proposed will advance our understanding of the function of the CNNM proteins in divalent cation transport and their regulation by Mg2+·ATP binding.