1.A.26 The Mg²⁺ Transporter-E (MgtE) Family

Several functionally characterized members of the MgtE/SLC41 family have been described. These include MgtE from the Gram-positive bacterium, Bacillus firmus OF4, and MgtE from the Gram-negative bacterium, Providencia stuartii. These proteins are capable of transporting Mg²⁺ and Co²⁺ but not Ni²⁺. The K_M values for Mg²⁺ and Co²⁺ uptake are in the 50-100 μM range. An MgtE homologue from Aeromonas hydrophila expressed in a Salmonella typhimurium strain that is deleted for all Mg²⁺ transporters complements the defect, both phenotypically and by allowing ⁵⁷Co²⁺ uptake (Merino et al., 2001). MstE (1.A.26.1.2), CLC (2.A.49.6.1) and HlyC/CorC (HCC; 9.A.40.1.2) may all share a hydrophilic domain.  The metal-binding site of the MgtE TM domain binds to Mg²⁺ approximately 500-fold more strongly than to Ca²⁺. Teng et al. 2022 determined the crystal structure of the MgtE TM domain in complex with Ca²⁺ at 2.5 Å resolution, revealing hexahydrated Ca²⁺. These results provide mechanistic insights into the ion selectivity of MgtE for Mg²⁺ over Ca²⁺.

MgtE is a homodimeric Mg²⁺-selective channel and is negatively regulated by high intracellular Mg²⁺ concentrations where the cytoplasmic domain of MgtE acts as a Mg²⁺ sensor, and Mg²⁺-dependent gating is dependent on lipid-protein interactions (Chatterjee et al. 2020). MgtE structures in the Mg²⁺-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg²⁺ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg²⁺ homeostasis. Jin et al. 2021 determined the cryo-EM structure of a MgtE-Fab complex in the absence of Mg²⁺ ions. The Mg²⁺-free MgtE TM domain structure and its comparison with the Mg²⁺-bound, closed-state structure, together with functional analyses, showed the Mg²⁺-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TMS2 and TMS5 helices at the glycine residues, which are important for channel activity (Jin et al. 2021).

Members of the family have been sequenced from a wide variety of bacteria, archaea and eukaryotes. Their phylogeny roughly follows that of the organismal taxonomies. They have sizes that vary considerably from 311 residues for the Methanococcus thermoautotrophicum protein, 463 residues for the Synechocystis homologue, and 513 residues for the human homologue, SLC41A1. The B. firmus transporter and several homologues examined have strongly charged, hydrophilic N-terminal domains (cytoplasmic) followed by a hydrophobic C-terminal domain with 5 putative transmembrane α-helical spanners. A central 100 residues resembles archaeal inositol monophosphate dehydrogenases (Kehres and Maguire, 2002). Kehres and Maguire (2002) suggested that the MgtE proteins are secondary carriers (uniporters?) with inwardly directed polarity. Hattori et al. (2009) have considered MgtE to function by a channel mechanism. They provided evidence that the MgtE cytosolic domain acts as a Mg²⁺ sensor to regulate gating of the pore in response to the intracellular Mg²⁺ concentration. This produces a mechanism for the maintenance of homeostasis conditions (Hattori et al., 2009).  Structure, mechanism and regulation have been reviewed (Payandeh et al. 2013; Sahni and Scharenberg 2013).

Mg²⁺ has the largest hydrated radius among all cations although its ionic radius is the smallest. How do Mg²⁺ transporters selectively recognize and dehydrate the large, fully hydrated Mg²⁺ cation for transport? Human homologues have been functionally characterized and suggested to be involved in magnesium homeostasis. Hattori et al. (2007) determined the crystal structures of the full-length Thermus thermophilus MgtE at 3.5 Å resolution, and of the cytosolic domain in the presence and absence of Mg²⁺ at 2.3 Å and 3.9 Å resolutions, respectively. The transporter adopts a homodimeric architecture. The carboxy-terminal domain has five TMSs. The amino-terminal cytosolic part consists of the superhelical N domain and tandemly repeated cystathionine-β-synthase domains. A solvent-accessible pore nearly traverses the transmembrane domains, with one potential Mg²⁺ bound to the conserved Asp 432 within the pore. The transmembrane helices #5 from both subunits close the pore through interactions with the 'connecting helices', which connect the cystathionine-β-synthase and transmembrane domains. Four putative Mg²⁺ ions are bound at the interface between the connecting helices and the other domains, and this may lock the closed conformation of the pore. A structural comparison of the two states of the cytosolic domains showed the Mg²⁺-dependent movement of the connecting helices, which might reorganize the transmembrane helices to open the pore. These findings suggest a homeostatic mechanism, in which Mg²⁺, bound between cytosolic domains, regulates Mg²⁺ flux by sensing the intracellular Mg²⁺ concentration. Whether this presumed regulation controls gating of an ion channel or opening of a secondary active transporter remains to be determined.

The cytosolic domain of MgtE undergoes a Mg²⁺-dependent structural change, which may gate the ion-conducting pore passing through the transmembrane domain. Ishitani et al. (2008) performed molecular dynamics simulations of the MgtE cytosolic domain which reproduced the structural changes of the cytosolic domain upon binding or releasing Mg²⁺. The roles of the N and CBS domains in the cytosolic domain and their respective Mg²⁺ binding sites were proposed.

Maruyama et al. (2012) showed that MgtE exhibits the channel-like electrophysiological property, i.e., Mg²⁺ transport occurs in accordance to the electrochemical potential of Mg²⁺. The Mg²⁺-permeation pathway opens in response to a decrease of the intracellular Mg²⁺ concentration, while it is completely closed at the intracellular Mg²⁺ concentration of 10 mM. The crystal structures of the MgtE dimer reveals that the Mg²⁺-sensing cytoplasmic region consists of the N and CBS domains. The Mg²⁺-bound state adopts a compact, globular conformation, which is stabilized by the coordination of a number of Mg²⁺ ions between these domains. On the other hand, in the Mg²⁺-unbound state, these domains are far apart, and fixed by the crystal packing. Maruyama et al. (2012) reported the backbone resonance assignments of the dimer of the cytoplasmic region of the MgtE from Thermus thermophilus with a molecular weight of 60 KDa, in the Mg²⁺-unbound state. 

The crystal structures of bacterial CorA and MgtE Mg²⁺ channels revealed that both structures are unique, unlike that of any other channel or transporter (Moomaw and Maguire 2008). Although structurally quite different, both CorA and MgtE appear to be gated in a similar manner through multiple Mg²⁺ binding sites in the cytosolic domain of the channels. These sites essentially serve as Mg²⁺ 'sensors' of cytosolic Mg²⁺ concentration. MgtE tightly regulates the intracellular Mg²⁺ concentration. The dimeric structure is composed of a total of ten transmembrane α-helices, forming a central pore. Intracellular soluble domains constitute the Mg²⁺ sensor. The ion selectivity for Mg²⁺ over Ca²⁺ resides in a central cavity in the transmembrane pore involving a conserved aspartate residue (Asp432) from each monomer. Two different classes of binding sites are present in MgtE: a high affinity site for Mg²⁺ (Kd 0.3 mM) with low Ca²⁺ affinity (Kd 80 mM), and a medium affinity site for Mg²⁺ (Kd 2 mM) and Ca²⁺ (Kd 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively (Kimura et al. 2018).

The transport reaction catalyzed by MgtE proteins is:

Mg²⁺ (or Co²⁺) (out) → Mg²⁺ (or Co²⁺) (in).

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