Crystal structure of the CorA Mg2+ transporter

Frontiers in physiology, 2014

Connexin channels mediate electrical coupling, intercellular molecular signaling, and extracellular release of signaling molecules. Connexin proteins assemble intracellularly as hexamers to form plasma membrane hemichannels. The docking of two hemichannels in apposed cells forms a gap junction channel that allows direct electrical and selective cytoplasmic communication between adjacent cells. Hemichannels and junctional channels are gated by voltage, but extracellular Ca (2+) also gates unpaired plasma membrane hemichannels. Unlike other ion channels, connexin channels do not contain discrete voltage- or Ca (2+)-sensing modules linked to a separate pore-forming module. All studies to date indicate that voltage and Ca (2+) sensing are predominantly mediated by motifs that lie within or are exposed to the pore lumen. The sensors appear to be integral components of the gates, imposing an obligatory structural linkage between sensing and gating not commonly present in other ion channel...

Cells

Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties up...

Metallomics

The biological chemistry of the transition metal ''transportome'' of Cupriavidus metallidurans Dietrich H. Nies This review tries to illuminate how the bacterium Cupriavidus metallidurans CH34 is able to allocate essential transition metal cations to their target proteins although these metals have similar charge-to-surface ratios and chemical features, exert toxic effects, compete with each other, and occur in the bacterial environment over a huge range of concentrations and speciations. Central to this ability is the ''transportome'', the totality of all interacting metal import and export systems, which, as an emergent feature, transforms the environmental metal content and speciation into the cellular metal mélange. In a kinetic flow equilibrium resulting from controlled uptake and efflux reactions, the periplasmic and cytoplasmic metal content is adjusted in a way that minimizes toxic effects. A central core function of the transportome is to shape the metal ion composition using high-rate and low-specificity reactions to avoid time and/or energy-requiring metal discrimination reactions. This core is augmented by metal-specific channels that may even deliver metals all the way from outside of the cell to the cytoplasm. This review begins with a description of the basic chemical features of transition metal cations and the biochemical consequences of these attributes, and which transition metals are available to C. metallidurans. It then illustrates how the environment influences the metal content and speciation, and how the transportome adjusts this metal content. It concludes with an outlook on the fate of metals in the cytoplasm. By generalization, insights coming from C. metallidurans shed light on multiple transition metal homoeostatic mechanisms in all kinds of bacteria including pathogenic species, where the ''battle'' for metals is an important part of the host-pathogen interaction.

Metallomics

The beta-proteobacterium Cupriavidus metallidurans is able to grow in metal-contaminated environments due to having sophisticated metal efflux systems. Here, the contribution of all seven known secondary metal uptake systems (ZupT, PitA, CorA 1 , CorA 2 , CorA 3 , ZntB, HoxN) to metal resistance is characterized. In a strategic deletion approach, all ten double deletion mutants, a variety of triple and quadruple mutants, and from the D4 mutant (DzupT DcorA 1 DcorA 2 DcorA 3) the mutants D5 (=D4 DpitA), D6 (=D4 DpitA DzntB), and finally D7 (DzupT DcorA 1 DcorA 2 DcorA 3 DpitA DzntB DhoxN) were constructed. Metal resistance, metal content, and regulation of expression of these genes were characterized in these mutants. The DzupT single deletion strain exhibited an extended lag phase in Tris-buffered liquid mineral salts medium (TMM) compared to its parent strain AE104, indicating a decreased fitness level. Further deletions up to D6 did not influence growth in TMM without added metals but fitness of the D7 strain dropped to a lower level compared to D6, D5 and DzupT. The cells of the D7 multiple deletion strain still contained all essential metals, demonstrating that additional metal import systems must exist in C. metallidurans. PitA was an important contributor of metal:phosphate complexes to C. metallidurans. Up to D5 no evidence was found for increased expression of the transporter genes to recruit substitutes for the deleted importers. Only the hoxN-lacZ reporter gene fusion displayed a changed expression pattern in the D6 strain, indicating recruitment of HoxN. Metal resistance of the deletion strains decreased along the deletion series although all strains still contained metal efflux systems: up to the D6 mutant the overall fitness was kept at the DzupT mutant strain level at the cost of a diminished competence to handle mM concentrations of transition metals. Together, these data demonstrated an important contribution of the seven secondary metal import systems to metal homeostasis in this bacterium.

Proceedings of the National Academy of Sciences, 2012

Magnesium ions (Mg 2+ ) are essential for life, but the mechanisms regulating their transport into and out of cells remain poorly understood. The CorA-Mrs2-Alr1 superfamily of Mg 2+ channels represents the most prevalent group of proteins enabling Mg 2+ ions to cross membranes. Thermotoga maritima CorA (TmCorA) is the only member of this protein family whose complete 3D fold is known. Here, we report the crystal structure of a mutant in the presence and absence of divalent ions and compare it with previous divalent ion-bound TmCorA structures. With Mg 2+ present, this structure shows binding of a hydrated Mg 2+ ion to the periplasmic Gly-Met-Asn (GMN) motif, revealing clues of ion selectivity in this unique channel family. In the absence of Mg 2+ , TmCorA displays an unexpected asymmetric conformation caused by radial and lateral tilts of protomers that leads to bending of the central, pore-lining helix. Molecular dynamics simulations support these movements, including a bell-like d...

Encyclopedia, 2021

Metal ions play several major roles in proteins: structural, regulatory, and enzymatic. The binding of some metal ions increase stability of proteins or protein domains. Some metal ions can regulate various cell processes being first, second, or third messengers. Some metal ions, especially transition metal ions, take part in catalysis in many enzymes. From ten to twelve metals are vitally important for activity of living organisms: sodium, potassium, magnesium, calcium, manganese, iron, cobalt, zinc, nickel, vanadium, molybdenum, and tungsten. This short review is devoted to structural, physical, chemical, and physiological properties of proteins, which specifically bind these metal cations.

Journal of Biological Chemistry, 2011

CorA is a family of divalent cation transporters ubiquitously present in bacteria and archaea. Although CorA can transport both Mg2+ and Co2+ almost equally well, its main role has been suggested to be that of primary Mg2+ transporter of prokaryotes and hence the regulator of Mg2+ homeostasis. The reason is that the affinity of CorA for Co2+ is relatively low and thus considered non-physiological. Here, we show that Thermotoga maritima CorA (TmCorA) is incapable of regulating the Mg2+ homeostasis and therefore cannot be the primary Mg2+ transporter of T. maritima. Further, our in vivo experiments confirm that TmCorA is a highly selective Co2+ transporter, as it selects Co2+ over Mg2+ at >100 times lower concentrations. In addition, we present data that show TmCorA to be extremely thermostable in the presence of Co2+. Mg2+ could not stabilize the protein to the same extent, even at high concentrations. We also show that addition of Co2+, but not Mg2+, specifically induces structur...

Metallomics, 2013

The highly conserved G-M-N motif of the CorA-Mrs2-Alr1 family of Mg 2+ channels has been shown to be essential for Mg 2+ transport. We performed random mutagenesis of the G-M-N sequence of Saccharomyces cerevisiae Mrs2p in an unbiased genetic screen. A large number of mutants still capable of Mg 2+ influx, albeit below the wild-type level, were generated. Growth complementation assays, performed in media supplemented with Ca 2+ or Co 2+ or Mn 2+ or Zn 2+ at varying concentrations, lead to identification of mutants with reduced growth in the presence of Mn 2+ and Zn 2+. We hereby conclude that (1) at least two, but predominantly all three amino acids of the G-M-N motif must be replaced by certain combinations of other amino acids to remain functional, (2) replacement of any single amino acid within the G-M-N motif always impairs the function of Mrs2p, and (3) we show that the G-M-N motif determines ion selectivity, likely in concurrence with the negatively charged loop at the entrance of the channel thereby forming the Mrs2p selectivity filter.

Cell Research, 2013

The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu 2+-and Ni 2+-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co 2+. Indeed, the structure of TtNikM2 containing a bound Co 2+ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.

Nature communications, 2014

Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg(2+) channel distributed in all domains of life that contributes to the maintenance of cellular Mg(2+) homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg(2+), Mn(2+) and Ca(2+). The high-resolution Mg(2+)-bound crystal structure clearly visualized the hydrated Mg(2+) ion within its selectivity filter. Based on those structures and biochemical analyses, we propose a cation selectivity mechanism for MgtE in which the geometry of the hydration shell of the fully hydrated Mg(2+) ion is recognized by the side-chain carboxylate groups in the selectivity filter. This is in contrast to the K(+)-selective filter of KcsA, which recognizes a dehydrated K(+) ion. Our results further revealed a cation-binding site on the periplasmic side, which regulate channel opening and prevents conduction of near-cognate ca...

PLoS Computational Biology, 2010

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, falsepositive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users.

Nature Structural & Molecular Biology, 2009

Zinc transporters play critical roles in cellular zinc homeostatic control. The 2.9-Å resolution structure of the zinc transporter YiiP from Escherichia coli reveals a richly charged dimerinterface stabilized by zinc binding. Site-directed fluorescent resonance energy transfer (FRET) measurements and mutation-activity analysis suggest that zinc binding triggers hinge movements of two electrically repulsive cytoplasmic domains pivoting around four salt-bridges situated at the juncture of the cytoplasmic and transmembrane domains. These highly conserved salt-bridges interlock transmembrane helices at the dimer-interface, well positioned to transmit zinc-induced inter-domain movements to reorient transmembrane helices, thereby modulating coordination geometry of the active-site for zinc transport. The cytoplasmic domain of YiiP is a structural mimic of metal trafficking proteins and the metal-binding domains of metal-transporting P-type ATPases. The use of this common structural module to regulate metal coordination chemistry may enable a tunable transport activity in response to cytoplasmic metal fluctuations. Zinc is the second most abundant transition metal in cells with a zinc quota in the submillimolar range. It is an essential element for cell growth and development1,2, yet excess zinc is highly cytotoxic3. Cellular zinc is largely associated with zinc-binding proteins, so that the free zinc concentration in the cytoplasm can be kept below the picomolar range to minimize its cytotoxic effects4. To provide ample supplies of cellular zinc and avoid excessive zinc accumulation at the same time, two distinct families of zinc transporters contribute to a delicate balance between zinc acquisition and removal. The ZRT, IRT-like proteins (ZIPs)5,6 and the cation diffusion facilitators (CDFs)7,8 facilitate zinc uptake into and efflux from the cytoplasm, respectively. Both ZIPs and CDFs are conserved in all living organisms from bacteria to plants and animals. Mammalian ZIPs form family 39 in the classification of solute carrier proteins (SLC39), while mammalian CDFs, also known as zinc transporters (ZnTs), belong to the SLC30 family. These zinc transporters utilize transmembrane electrochemical potentials to drive Zn(II) transport across the membrane barrier in opposite directions. Among them, CDFs function as Zn(II)/proton antiporters9,10. Zinc-dependent expression of complementary zinc transporters is thought to control steadystate equilibrium of zinc fluxes around a homeostatic set point11. On the other hand, abrupt zinc overloading may demand a fast-responding regulatory mechanism to prevent toxic zinc Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The Journal of Chemical Physics, 2016

Structure of CorA protein and its inner (i.corA) and outer (o.corA) transmembrane (TM) components are investigated as a function of temperature by a coarse-grained Monte Carlo simulation. Thermal response of i.corA is found to differ considerably from that of the outer component, o.corA. Analysis of the radius of gyration reveals that the inner TM component undergoes a continuous transition from a globular conformation to a random coil structure on raising the temperature. In contrast, the outer transmembrane component exhibits an abrupt (nearly discontinuous) thermal response in a narrow range of temperature. Scaling of the structure factor shows a globular structure of i.corA at a low temperature with an effective dimension D ∼ 3 and a random coil at a high temperature with D ∼ 2. The residue distribution in o.corA is slightly sparser than that of i.corA in a narrow thermos-responsive regime. The difference in thermos-response characteristics of these components (i.corA and o.corA...

Life science alliance, 2023

Mitochondrial RNA splicing 2 (MRS2) forms a magnesium (Mg 2+) entry protein channel in mitochondria. Whereas MRS2 contains two transmembrane domains constituting a pore on the inner mitochondrial membrane, most of the protein resides within the matrix. Yet, the precise structural and functional role of this obtrusive amino terminal domain (NTD) in human MRS2 is unknown. Here, we show that the MRS2 NTD self-associates into a homodimer, contrasting the pentameric assembly of CorA, an orthologous bacterial channel. Mg 2+ and calcium suppress lower and higher order oligomerization of MRS2 NTD, whereas cobalt has no effect on the NTD but disassembles full-length MRS2. Mutating-pinpointed residues-mediating Mg 2+ binding to the NTD not only selectively decreases Mg 2+-binding affinity~sevenfold but also abrogates Mg 2+ binding-induced secondary, tertiary, and quaternary structure changes. Disruption of NTD Mg 2+ binding strikingly potentiates mitochondrial Mg 2+ uptake in WT and Mrs2 knockout cells. Our work exposes a mechanism for human MRS2 autoregulation by negative feedback from the NTD and identifies a novel gain of function mutant with broad applicability to future Mg 2+ signaling research.