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.     

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A,Mg2+ Ap5A,and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap₅A, Mn²⁺ Ap₅A, and Mg²⁺ Ap₅A have been determined by X-ray crystallography to resolutions of 1.6 Å, 1.85 Å, and 1.96 Å, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap₅A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn²⁺ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 Å. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg²⁺ Ap₅A and Mn²⁺ Ap₅A structures, Mg²⁺ and Mn²⁺ demonstrate six coordinate octahedral geometry. The interactions of the Mg²⁺-coordinated water molecules with the protein and Ap₅A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for β-γ (preferred by Mg²⁺) rather than α-γ (preferred by Mn²⁺) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation. Proteins 32:276–288, 1998. © 1998 Wiley-Liss, Inc.     

The aquaporin MePIP2;7 improves MeMGT9-mediated Mg2+ acquisition in cassava

Aquaporins are important transmembrane water transport proteins which transport water and several neutral molecules. However, how aquaporins are involved in the synergistic transport of Mg²⁺and water remains poorly understood. Here, we found that the cassava aquaporin Me PIP2;7 was involved in Mg²⁺transport through interaction with Me MGT9, a lower affinity magnesium transporter protein. Knockdown of Me PIP2;7 in cassava led to magnesium deficiency in basal mature leaves wi...     

Fluoride inhibition of yeast enolase: Crystal structure of the enolase–Mg2+–F−–Pi complex at 2.6 Å resolution

Enolase in the presence of its physiological cofactor Mg²⁺ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase–Mg²⁺–F^?–P_i has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate–enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg²⁺ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg²⁺. The synergism of inhibition by F^? and P_i most probably is due to the requirement of P_i, for the closed conformation. It is possible that other Mg²⁺-dependent enzymes that have OH^? ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.     

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.     

Transmembrane Mg2+ Currents and Intracellular Free Mg2+ Concentration in Paramecium tetraurelia

The properties of Mg²⁺ conductances in Paramecium tetraurelia were investigated under two-electrode voltage clamp. When bathed in physiological Mg²⁺ concentrations (0.5 mm), depolarizing steps from rest elicited a prominent Mg²⁺-specific current (I _Mg) that has been noted previously. The dependence of this current on extracellular Mg²⁺ approximated that of Mg²⁺-induced backward swimming, demonstrating that I _Mg contributes to normal membrane excitation and behavior in this ciliate. Closer analysis revealed that the Mg²⁺ current deactivated biphasically. While this might suggest the involvement of two Mg²⁺-specific pathways, both tail-current components were affected similarly by current-specific mutations and they had similar ion selectivities, suggesting a common pathway. In contrast, a Mg²⁺ current activated upon hyperpolarization could be separated into three components. The first, I _Mg, had similar properties to the current activated upon depolarization. The second was a nonspecific divalent cation current (I _NS) that was revealed following suppression of I _Mg by eccentric mutation. The final current was relatively minor and was revealed following suppression of I _Mg and I _NS by obstinate A gene mutation. Reversal-potential analyses suggested that I _Mg and I _NS define two intracellular compartments that contain, respectively, low (0.4 mm) and high (8 mm) concentrations of Mg²⁺. Measurement of intracellular free Mg²⁺ using the fluorescent dye, Mag-fura-2, suggested that bulk [Mg²⁺] _i rests at around 0.4 mm in Paramecium. Received: 12 January 1998/Revised: 16 March 1998     

Luminescence properties of red‐emission Mg4Nb2O9:Eu3+ phosphor

Red‐emitting Mg₄Nb₂O₉:Eu³⁺ phosphor is synthesized via a solid‐state reaction method in air, and its crystal structure and luminescence are investigated. The phosphor can be excited efficiently by ~ 395 nm light, coupled well with a ~ 395 nm near‐ultraviolet chip and emits red light at ~ 613 nm with sharp spectra due to ⁵D₀ → ⁷ F₂ transition of the Eu³⁺ ion. Mg₄Nb₂O₉:Eu³⁺ phosphor sintered at 1350 ºC shows Commission international de I'Eclairage (CIE) chromaticity coordinates of x = 0.6354, y = 0.3592, and is a potential red‐emitting phosphor candidate for white light‐emitting diodes (W‐LEDs) under ~ 395 nm near‐ultraviolet LED chip excitation. Copyright © 2014 John Wiley & Sons, Ltd.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.     

DNA aggregation induced by Mg2+ ions under different conditions

Cations‐induced DNA aggregation can modify the local structure of oligonucleotides and has potential applications in medicine and biotechnology. Here, we used atomic force microscopy to investigate λ‐DNA aggregation on Mg²⁺‐treated glass (Mg²⁺/glass) and in Mg²⁺ solution. Atomic force microscopy topography images showed that some DNA fragments were slightly stacked together on 10 mM Mg²⁺/glass and stacked stronger on ≥50 mM Mg²⁺/glass. They also showed that DNA aggregated stronger in Mg²⁺ solution than on Mg²⁺/glass, ie, DNAs are strongly stacked and twisted at 10 mM Mg²⁺, rolled together at 50 mM Mg²⁺, and slightly aggregated to form small particles at 100 mM Mg²⁺. At a specific condition, ie, heating λ‐DNA to 92°C, cooling down to 75°C, adding Mg²⁺, and vortexing the resulting solution, DNA strongly aggregated and formed pancake‐like shapes at 10 and 50 mM or a large aggregate at 100 mM Mg²⁺ solutions. Our results may be helpful for medical applications and gene therapy using cation‐DNA technology.     

The influence of Mg2+ coordination on 13C and 15N chemical shifts in CKI1RD protein domain from experiment and molecular dynamics/density functional theory calculations

Sequence dependence of ¹³C and ¹⁵N chemical shifts in the receiver domain of CKI1 protein from Arabidopsis thaliana, CKI1_RD, and its complexed form, CKI1_RD?Mg²⁺, was studied by means of MD/DFT calculations. MD simulations of a 20–ns production run length were performed. Nine explicitly hydrated structures of increasing complexity were explored, up to a 40‐amino‐acid structure. The size of the model necessary depended on the type of nucleus, the type of amino acid and its sequence neighbors, other spatially close amino acids, and the orientation of amino acid NH groups and their surface/interior position. Using models covering a 10 and a 15 Å environment of Mg²⁺, a semi‐quantitative agreement has been obtained between experiment and theory for the V67?I73 sequence. The influence of Mg²⁺ binding was described better by the 15 Å as compared to the 10 Å model. Thirteen chemical shifts were analyzed in terms of the effect of Mg²⁺ insertion and geometry preparation. The effect of geometry was significant and opposite in sign to the effect of Mg²⁺ binding. The strongest individual effects were found for ¹⁵N of D70, S74, and V68, where the electrostatics dominated; for ¹³Cβ of D69 and ¹⁵N of K76, where the influences were equal, and for ¹³Cα of F72 and ¹³Cβ of K76, where the geometry adjustment dominated. A partial correlation between dominant geometry influence and torsion angle shifts upon the coordination has been observed. Proteins 2016; 84:686–699. © 2016 Wiley Periodicals, Inc.     

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.     

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

Mrs2p forms a high conductance Mg2+ selective channel in mitochondria

Members of the CorA-Mrs2-Alr1 superfamily of Mg²⁺ transporters are ubiquitous among pro- and eukaryotes. The crystal structure of a bacterial CorA protein has recently been solved, but the mode of ion transport of this protein family remained obscure. Using single channel patch clamping we unequivocally show here that the mitochondrial Mrs2 protein forms a Mg²⁺-selective channel of high conductance (155 pS). It has an open probability of ∼60% in the absence of Mg²⁺ at the matrix site, which decreases to ∼20% in its presence. With a lower conductance (∼45 pS) the Mrs2 channel is also permeable for Ni²⁺, whereas no permeability has been observed for either Ca²⁺, Mn²⁺, or Co²⁺. Mutational changes in key domains of Mrs2p are shown either to abolish its Mg²⁺ transport or to change its characteristics toward more open and partly deregulated states. We conclude that Mrs2p forms a high conductance Mg²⁺ selective channel that controls Mg²⁺ influx into mitochondria by an intrinsic negative feedback mechanism.     

Distances between the functional sites of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase and the lipid/water interface

Measurements of fluorescence energy transfer have been performed to determine the distance between the lipid-water interface and the ATP-binding site in the (Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum. The calculated distance between the donor, FITC bound to the protein (nucleotide binding-site marker), and the acceptor, rhodamine-5′-isothiocyanyldipalmitoylphosphatidylethanolamine (RITC-DPPE) incorporated in the membrane, was in the range of 34–42 Å. In addition the distance between the high affinity Ca²⁺-binding sites and the lipid/water interface has been calculated by luminescence energy transfer from Tb³⁺ bound to the Ca²⁺ sites to RITC-DPPE included in the membrane, and it was approx. 10 Å.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Crystallization and preliminary diffraction analysis of Ca2+-calmodulin-drug and apocalmodulin-drug complexes

Ca²⁺-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3”-(β-chloroethyl)-2”,4”-dioxo-3,5”-spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4-di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 Å and differ in unit cell parameters from each other as well as from crystals of Ca²⁺-calmodulin or Ca²⁺-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca²⁺-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca²⁺-free calmodulin crystals diffracting beyond 2.5 Å resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca²⁺-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies. © 1997 Wiley-Liss Inc.     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

Net Mg2+ uptake in relation to the amount of exchangeable Mg2+ in the Donnan free space of ryegrass roots

Ammonium acetate and BaCl₂-triethanolamine were used to desorb Mg²⁺ from the root Donnan free space (DFS) of 23-d-old ryegrass (Lolium multiflorum Lam. cvs. Gulf and Wilo). Amounts of desorbed Mg²⁺ increased with the increase in Mg²⁺ activity of the nutrient solution. Slightly less Mg²⁺ was desorbed by Ba²⁺ than by NH₄ ⁺. Previously published data on short-term net Mg²⁺ uptake by intact 23-d-old ryegrass plants of the two cultivars were linearly related to the amount of exchangeable Mg⁺ desorbed from the root DFS (r²=0.90 and 0.81 for the desorption by NH₄ ⁺ and Ba²⁺, respectively). A sward of Mg²⁺ ions attracted to the negative charges of the cell surface is suggested to represent a part of a pool of Mg²⁺ available for active transport through the plasmalemma.