The structure and regulation of magnesium selective ion channels

The magnesium ion (Mg^2 +) is the most abundant divalent cation within cells. In man, Mg^2 +-deficiency is associated with diseases affecting the heart, muscle, bone, immune, and nervous systems. Despite its impact on human health, little is known about the molecular mechanisms that regulate magnesium transport and storage. Complete structural information on eukaryotic Mg^2 +-transport proteins is currently lacking due to associated technical challenges. The prokaryotic MgtE and CorA magnesium transport systems have recently succumbed to structure determination by X-ray crystallography, providing first views of these ubiquitous and essential Mg^2 +-channels. MgtE and CorA are unique among known membrane protein structures, each revealing a novel protein fold containing distinct arrangements of ten transmembrane-spanning α-helices. Structural and functional analyses have established that Mg^2 +-selectivity in MgtE and CorA occurs through distinct mechanisms. Conserved acidic side-chains appear to form the selectivity filter in MgtE, whereas conserved asparagines coordinate hydrated Mg^2 +-ions within the selectivity filter of CorA. Common structural themes have also emerged whereby MgtE and CorA sense and respond to physiologically relevant, intracellular Mg^2 +-levels through dedicated regulatory domains. Within these domains, multiple primary and secondary Mg^2 +-binding sites serve to staple these ion channels into their respective closed conformations, implying that Mg^2 +-transport is well guarded and very tightly regulated. The MgtE and CorA proteins represent valuable structural templates to better understand the related eukaryotic SLC41 and Mrs2–Alr1 magnesium channels. Herein, we review the structure, function and regulation of MgtE and CorA and consider these unique proteins within the expanding universe of ion channel and transporter structural biology.     

MgtC as a Horizontally-Acquired Virulence Factor of Intracellular Bacterial Pathogens: Evidence from Molecular Phylogeny and Comparative Genomics

MgtC is a virulence factor required for intramacrophage survival and growth in low Mg²⁺ medium in two pathogens that are not phylogenetically related, Salmonella typhimurium and Mycobacterium tuberculosis. In S. typhimurium, mgtC is carried by the SPI-3 pathogenicity island and hybridization studies have suggested that the distribution of mgtC among enterobacteria is limited. In the present study, we searched for the presence of mgtC-like sequences in eubacterial genomes. Analyses of MgtC-like proteins phylogeny and mgtC-like chromosomal context support the hypothesis that mgtC has been acquired by horizontal gene transfer repeatedly throughout bacterial evolution. In addition, the phylogenetic analysis revealed the existence of a subgroup of proteins, that includes the S. typhimurium and M. tuberculosis MgtC proteins, as well as MgtC-related proteins from other pathogens that are able to survive in macrophages, B. melitensis and Y. pestis. We propose that MgtC has a similar function in all these distantly related pathogens, most likely providing the ability to grow in a low Mg²⁺ environment. Present address: (Anne-Béatrice Blanc-Potard) Inserm U431, Faculté de Médecine, 30900 Nîmes, France     

Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems.

Three loci in Salmonella typhimurium (corA, mgtA, and mgtB) code for components of distinct Mg2+ transport systems (S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). Strains carrying one wild-type and two mutant alleles of the three loci were constructed to study the kinetics and specificity of ion transport of each system in isolation. The transport systems had different Km and Vmax values for Mg2+ uptake, and each was inhibited by other divalent cations in a distinct rank order of potency: for CorA, Mg2+ greater than Mn2+ greater than Co2+ greater than Ni2+ greater than Ca2+; for MgtA, Zn2+ greater than or equal to Mg2+ greater than Ni2+ approximately Co2+ greater than Ca2+; and for MgtB, Mg2+ approximately Ni2+ approximately Ni2+ greater than Mn2+ much greater than Ca2+. Other differences among the three systems were apparent. The CorA transport system functioned as a Mg2+-Mg2+ exchange system, mediating both efflux and influx of Mg2+. Neither the MgtA nor the MgtB system could mediate Mg2+ efflux. Transport via the MgtB system was very temperature sensitive; Mg2+ was transported at 37 degrees C but not at 20 degrees C. The MgtA and the MgtB transport systems were found to be regulated by the extracellular concentration of Mg2+.     

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters

Magnesium ions (Mg²⁺) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg²⁺ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg²⁺ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg²⁺ channels and transporters. In this review article, we provide an overview of the families of Mg²⁺ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.     

Functional Similarity between Archaeal and Bacterial CorA Magnesium Transporters

The constitutively expressed CorA Mg²⁺ transporter is the major Mg²⁺ influx system of Salmonella typhimurium and Escherichia coli. Genomic sequence data indicated the presence of a homolog in the archaeal organism Methanococcus jannaschii. The putative M. jannaschii CorA was expressed in an Mg²⁺-transport-deficient strain of S. typhimurium to determine its functional characteristics. The archaeal CorA homolog is a functional Mg²⁺ uptake system when expressed in S. typhimurium and has properties which are highly similar to those of the normal CorA transporter of S. typhimurium despite having a low level of sequence identity with the protein and being expressed in a lipid membrane of quite different composition than normal. This implies that the overall function of the proteins is the same and further suggests that their structures are very similar.     

Nickel resistance in fission yeast associated with the magnesium transport system

We isolated and characterized a nickel (Ni²⁺)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni²⁺ and Zn²⁺, but not to Cd²⁺, Co²⁺, and Cu²⁺. The growth rate of GA1 increased proportionally with increasing Mg²⁺ concentrations until 50 mM Mg²⁺. The GA1 mutation phenotype suggests a defect in Mg²⁺ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg²⁺ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro 153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni²⁺ resistance in fission yeast. Specifically, that reducing Mg²⁺ influx through the CorA Mg²⁺ transport membrane protein confers Ni²⁺ resistance in S. pombe. We also report that Ni²⁺ ion detoxification of the fission yeast is related to histidine metabolism and pH.     

Structure and electrostatic property of cytoplasmic domain of ZntB transporter

ZntB is the distant homolog of CorA Mg²⁺ transporter within the metal ion transporter superfamily. It was early reported that the ZntB from Salmonella typhimurium facilitated efflux of Zn²⁺ and Cd²⁺, but not Mg²⁺. Here, we report the 1.90 Å crystal structure of the intracellular domain of ZntB from Vibrio parahemolyticus. The domain forms a funnel-shaped homopentamer that is similar to the full-length CorA from Thermatoga maritima, but differs from two previously reported dimeric structures of truncated CorA intracellular domains. However, no Zn²⁺ or Cd²⁺ binding sites were identified in the high-resolution structure. Instead, 25 well-defined Cl⁻ ions were observed and some of these binding sites are highly conserved within the ZntB family. Continuum electrostatics calculations suggest that the central pore of the funnel is highly attractive for cations, especially divalents. The presence of the bound Cl⁻ ions increases the stability of cations along the pore suggesting they could be important in enhancing cation transport.     

Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed Tryptophan Fluorescence

Magnesium is the most abundant divalent cation present in the cell, and an abnormal Mg²⁺ homeostasis is associated with several diseases in humans. However, among ion channels, the mechanisms of intracellular regulation and transport of Mg²⁺ are poorly understood. MgtE is a homodimeric Mg²⁺-selective channel and is negatively regulated by high intracellular Mg²⁺ concentration where the cytoplasmic domain of MgtE acts as a Mg²⁺ sensor. Most of the previous biophysical studies on MgtE have been carried out in detergent micelles and the information regarding gating-related structural dynamics of MgtE in physiologically-relevant membrane environment is scarce. In this work, we monitored the changes in gating-related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in micelles and membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain site-specific information on the gating-related structural dynamics of MgtE in membrane-mimetic systems. Our results indicate that Mg²⁺-induced gating might involve the possibility of a ‘conformational wave’ from the cytosolic N-domain to transmembrane domain of MgtE. Although MgtE is responsive to Mg²⁺-induced gating in both micelles and membranes, the organization and dynamics of MgtE is substantially altered in physiologically important phospholipid membranes compared to micelles. This is accompanied by significant changes in hydration dynamics and conformational heterogeneity. Overall, our results highlight the importance of lipid-protein interactions and are relevant for understanding gating mechanism of magnesium channels in general, and MgtE in particular.     

The structure of MgtE in the absence of magnesium provides new insights into channel gating

MgtE is a Mg²⁺ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg²⁺ homeostasis. The previously determined 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. There are no structures of the transmembrane (TM) domain for MgtE in Mg²⁺-free conditions, and the pore-opening mechanism has thus remained unclear.Here, we determined the cryo-electron microscopy (cryo-EM) structure of the 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 TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

MgtE is a magnesium-selective ion channel whose gating is regulated by cytoplasmic magnesium concentration; this cryo-EM study reveals how MgtE undergoes magnesium-dependent structural changes to open the pore on the cytoplasmic side.     

Transport of Magnesium by a Bacterial Nramp-Related Gene

Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5–2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.     

Backbone resonance assignments for the cytoplasmic region of the Mg2+ transporter MgtE in the Mg2+-unbound state

Magnesium ion (Mg²⁺) is an essential metal element for life, and has many cellular functions, including ATP utilization, activation of enzymes, and maintenance of genomic stability. The intracellular Mg²⁺ concentration is regulated by a class of transmembrane proteins, called Mg²⁺ transporters. One of the prokaryotic Mg²⁺ transporters, MgtE, is a 450-residue protein, and functions as a dimer. We previously reported that MgtE exhibits the channel-like electrophysiological property, i.e., it permeates Mg²⁺ according to the electrochemical potential of Mg²⁺. The Mg²⁺-permeation pathway opens in response to the 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 revealed that the Mg²⁺-sensing cytoplasmic region consists of the N and CBS domains. The Mg²⁺-bound state of MgtE 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. Therefore, structural analyses in solution were awaited, in order to characterize the Mg²⁺-dependent alteration of the MgtE structure and dynamics relevant to its gating. In this paper, we report 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 homologous Arabidopsis MRS2/MGT/CorA-type Mg2+ channels,AtMRS2-10 and AtMRS2-1 exhibit different aluminum transport activity

Magnesium (Mg²⁺) plays a critical role in many physiological processes. The AtMRS2/MGT family, which consists of nine Arabidopsis genes (and two pseudo-genes) belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-10 and AtMRS2-1 possess the signature GlyMetAsn sequence conserved in the CorA superfamily; however, they have low sequence conservation with CorA. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-10 and AtMRS2-1 mediated rapid Mg²⁺ uptake into proteoliposomes. The rapid Mg²⁺ uptake through AtMRS2-10 was inhibited by aluminum. An assay using the Al-sensitive dye morin indicated Al uptake into the proteoliposomes through AtMRS2-10. AtMRS2-10 also exhibited Ni²⁺ transport activity but almost no Co²⁺ transport activity. The rapid Mg²⁺ uptake through AtMRS2-1 was not inhibited by aluminum. Al uptake into the proteoliposomes through AtMRS2-1 was not observed. The functional complementation assay in Escherichia coli strain TM2 showed that AtMRS2-1 was capable of mediating Mg²⁺ uptake. Heterologous expression using the E. coli mutant cells also showed that the E. coli cells expressing AtMRS2-1 was more resistant to aluminum than the E. coli cells expressing AtMRS2-10. The results suggested that AtMRS2-10 transported Al into the E. coli cells, and then the transported Al inhibited the growth of E. coli. AtMRS2-1 has been localized to the Arabidopsis tonoplast, indicating that AtMRS2-1 is exposed to much higher concentration of aluminum than AtMRS2-10. Under the conditions, it may be required that the Mg²⁺ transport of AtMRS2-1 is insensitive to Al inhibition, and AtMRS2-1 is impermeable to Al.     

Mg2+‐dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis

The MgtE family of Mg²⁺ transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full‐length MgtE and of its cytosolic domain in the presence and absence of Mg²⁺ suggested a Mg²⁺‐homeostasis mechanism, in which the MgtE cytosolic domain acts as a ‘Mg²⁺ sensor’ to regulate the gating of the ion‐conducting pore in response to the intracellular Mg²⁺ concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full‐length structure precluded an unambiguous characterization of these regulatory divalent‐cation‐binding sites. Here, we showed that MgtE is a highly Mg²⁺‐selective channel gated by Mg²⁺ and elucidated the Mg²⁺‐dependent gating mechanism of MgtE, using X‐ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg²⁺ homeostasis through cooperative Mg²⁺ binding to the MgtE cytosolic domain.     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

Functional reconstitution and characterization of the Arabidopsis Mg2+ transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg²⁺) plays critical role in many physiological processes. The mechanism of Mg²⁺ transport has been well documented in bacteria; however, less is known about Mg²⁺ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg²⁺ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg²⁺ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg²⁺ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg²⁺ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg²⁺ uptake. The AtMRS2-10-mediated Mg²⁺ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co²⁺ and Ni²⁺; however, it was not inhibited by Ca²⁺, Fe²⁺, or Fe³⁺. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al³⁺. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships.     

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

The CorA Mg²⁺ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg²⁺ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg²⁺ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg²⁺ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3?Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg²⁺ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of ⁶³Ni²⁺, indicating functionality. Thus, absence of Mg²⁺ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg²⁺ channel and not a Co²⁺ channel.