1.A.35 The CorA/Mrs2 Metal Ion Transporter (MIT) Family

The MIT (CorA/Mrs2) family, also called the CorA or Mrs2 family, is a large and diverse family with sequenced members in Gram-positive and Gram-negative bacteria, blue-green bacteria, archaea, plants, animals, yeast, slime molds, Guillardia theta, and Plasmodium. Functionally characterized proteins include the Mg²⁺-Co²⁺-Ni²⁺ CorA permeases of Salmonella typhimurium and E. coli, a Zn²⁺-Cd²⁺ effluxing system in S. typhimurium, a divalent cation transporting CorA homologue in Methanococcus janaschii, aluminum-resistance Mg²⁺ transport permeases (Al^R1p and Al^R2p) of Saccharomyces cerevisiae and a putative manganese-resistance magnesium transport permease (Mn^R2p) of S. cerevisiae. Most members of the MIT family are between 300 and 400 amino acyl residues in length and possess two (or three) putative transmembrane α-helical spanners (TMSs). It seems likely that some homologues have two and others have three TMSs. The greatest degree of conservation between homologues is found in TMSs 1 and 2 of the Thermotoga maritima protein. The yeast metal resistance proteins, which are 850-900 amino acyl residues in length, also exhibit two or three putative TMSs. Overexpression of the yeast proteins, Al^R1p and Mn^R2p, overcomes toxicity to aluminum and manganese, respectively.

The crystal structure of the CorA homologue from Thermotoga maritima has been solved at 3.9 Å resolution for the full-length protein and at 1.85 Å resolution for the cytoplasmic domain (Lunin et al., 2006). It is a funnel-shaped homopentamer with 2 TMSs per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg²⁺ ion was bound between monomers at a conserved site in the cytoplasmic domain suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg²⁺. These results contrast with those of Wang et al. (2006) who identified a soluble oligomeric N-terminal domain of the E. coli CorA that appeared to be tetrameric and to bind its substrates with the same affinity of native CorA (Wang et al., 2006).

The intracellular funnel domain of CorA constitutes an allosteric regulatory module that can be engineered to promote an activated or closed state (Payandeh et al., 2008). A periplasmic gate is centered about a proline-induced kink of the pore-lining helix. 'Helix-straightening' mutations result in gain-of-function. The narrowest constriction along the pore is a hydrophobic gate, likely forming an energetic barrier to ion flux. Highly conserved acidic residues found in the short periplasmic loop are not essential for CorA function or Mg²⁺ selectivity but may be required for proper protein folding and stability. Cation selectivity by the CorA Mg²⁺ channel requires a fully hydrated cation (Moomaw and Maguire, 2010).  The structure, mechanism and regulation of divalent ion transport via MIT family channels have been reviewed (Guskov and Eshaghi 2012; Payandeh et al. 2013).

The CorA permeases of S. typhimurium and E. coli mediate both influx and efflux of Mg²⁺. They transport Mg²⁺, Co²⁺ and Ni²⁺ but not Fe²⁺ (Papp and Maguire, 2004). Mg²⁺ is transported with an apparent K_M of 20-30 μM. The archaeal CorA protein is functionally similar to the CorA homologues of enteric bacteria. The yeast proteins appear to exhibit broad specificity transporting a wide range of di- and trivalent metal cations. In this respect, and also with respect to topology, MIT family members resemble channel proteins. The ZntB protein is a very distant homologue showing greatest similarity to the M. janaschii protein but catalyzing efflux of Zn²⁺ and Ca²⁺ (Worlock and Smith, 2002).

The CorA proteins of E. coli and S. typhimurium are each 316 amino acyl residues in length. Hydropathy analysis had predicted two transmembrane α-helical spanners (TMSs) in the C-terminal regions of these proteins. Site-specific mutagenesis studies of three conserved residues in TMS 3 suggest that they contribute to the Mg²⁺ transport pathway. In prokaryotes, cellular Mg²⁺ homeostasis is orchestrated via the CorA, MgtA/B, MgtE, and CorB/C Mg²⁺ transporters (Franken et al. 2022). For CorA, MgtE, and CorB/C, the motifs that form the selectivity pore, are conserved during evolution. Thus, CNNM proteins, the vertebrate orthologues of CorB/C, also have Mg²⁺ transport capacity. Whereas CorA and CorB/C proteins share the gross quaternary structure and functional properties with their respective orthologues, the MgtE channel only shares the selectivity pore with SLC41 Na⁺/Mg²⁺ transporters (Franken et al. 2022).

The transport mode and energy coupling mechanism(s) are poorly understood. A homopentameric structure for several CorA homologues has been established (Lunin et al., 2006), a channel-type mechanism rather than a carrier-type mechanism is operative, based on its high capacity and its 'gating' properties. CorA of S. typhimurium is a high capacity transporter that is constitutively synthesized. Some MIT family members may serve as the major Mg²⁺ influx systems in several prokaryotes, but others may catalyze divalent cation efflux.

Mrs2 of Saccharomyces cerevisiae is essential for the splicing of group II introns from RNA in mitochondria, and independently, for the maintenance of a functional respiratory system. Mrs2 is an integral protein of the inner mitochondrial membrane. It exhibits two adjacent putative TMSs in its C-terminal half. The large N-terminal domain and a shorter C-terminal domain are probably localized to the mitochondrial matrix. Mrs2 is distantly related to the E. coli CorA protein and other members of the MIT family. Null mutations in the mrs2 gene give mitochondria with low internal Mg²⁺ concentrations, and overexpression of the E. coli corA gene in yeast partially suppresses the mrs2 phenotype; CorA also partially restores intramitochondrial Mg²⁺ concentrations. More recently, it has been shown directly that Mrs2 is the electrophoretic mitochondrial Mg²⁺ uptake system, involved in Mg²⁺ homeostasis. It catalyzes Mg²⁺ uptake in the presence of a membrane potential and Mg²⁺ efflux in its absence (Kolisek et al., 2003). Thus, it functions like a Mg²⁺ channel as expected for a MIT family member. Mrs2 is demonstrably homologous to another S. cerevisiae ORF (YPL060w), as well as ORFs from other yeast, fungi, protozoans, plants and animals.

The transport reaction probably catalyzed by Mrs2 is:

Mg²⁺ (cytoplasm) Mg²⁺ (mitochondrial matrix)

The transport reaction generally catalyzed by MIT family members is:

M²⁺_(in) (or possible M³⁺_(in)) M²⁺_(out) (or possibly M³⁺_(out))
M²⁺ = a divalent heavy metal ion