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CorA is a Mg 2+ channel that plays a key role in the homeostasis of intracellular Mg 2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg 2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg 2+ , which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorA CD ). The Mg 2+ -binding sites of CorA CD differ depending on the species, suggesting that the Mg 2+ -binding modes and Mg 2+ -mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg 2+ -binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorA CD (cjCorA CD ). cjCorA CD adopts a three-layered α/β/α structure as observed in other CorA orthologs. Interestingly, cjCorA CD exhibited enhanced thermostability in the presence of Ca 2+ , Ni 2+ , Zn 2+ , or Mn 2+ in addition to Mg 2+ , indicating that cjCorA CD interacts with diverse divalent cations. This cjCorA CD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorA CD structure away from the pentamerization interface. Consistently, cjCorA CD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorA CD binds divalent cations in a unique pentamerization-independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Calcium and magnesium are the most abundant divalent cations in the body. The plasma level is controlled by coordinated interaction between intestinal absorption, reabsorption in the kidney, and, for calcium at least, bone storage and exchange. The kidney adjusts urinary excretion of these ions in response to alterations in their systemic concentration. Free ionized and anion-complexed calcium and magnesium are filtered at the glomerulus. The majority (i.e., >85%) of filtered divalent cations are reabsorbed via paracellular pathways from the proximal tubule and thick ascending limb (TAL) of the loop of Henle. Interestingly, the largest fraction of filtered calcium is reabsorbed from the proximal tubule (65%), while the largest fraction of filtered magnesium is reclaimed from the TAL (60%). The paracellular pathways mediating these fluxes are composed of tight junctional pores formed by claudins. In the proximal tubule, claudin-2 and claudin-12 confer calcium permeability, while the exact identity of the magnesium pore remains to be determined. Claudin-16 and claudin-19 contribute to the calcium and magnesium permeable pathway in the TAL. In this review, we discuss the data supporting these conclusions and speculate as to why there is greater fractional calcium reabsorption from the proximal tubule and greater fractional magnesium reabsorption from the TAL., (© 2022 New York Academy of Sciences.)

Magnesium (Mg 2+ ) is the most prevalent divalent intracellular cation. As co-factor in many enzymatic reactions, Mg 2+ is essential for protein synthesis, energy production, and DNA stability. Disturbances in intracellular Mg 2+ concentrations, therefore, unequivocally result in delayed cell growth and metabolic defects. To maintain physiological Mg 2+ levels, all organisms rely on balanced Mg 2+ influx and efflux via Mg 2+ channels and transporters. This review compares the structure and the function of prokaryotic Mg 2+ transporters and their eukaryotic counterparts. In prokaryotes, cellular Mg 2+ homeostasis is orchestrated via the CorA, MgtA/B, MgtE, and CorB/C Mg 2+ transporters. For CorA, MgtE, and CorB/C, the motifs that form the selectivity pore are conserved during evolution. These findings suggest that CNNM proteins, the vertebrate orthologues of CorB/C, also have Mg 2+ 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 + /Mg 2+ transporters. In eukaryotes, TRPM6 and TRPM7 Mg 2+ channels provide an additional Mg 2+ transport mechanism, consisting of a fusion of channel with a kinase. The unique features these TRP channels allow the integration of hormonal, cellular, and transcriptional regulatory pathways that determine their Mg 2+ transport capacity. Our review demonstrates that understanding the structure and function of prokaryotic magnesiotropic proteins aids in our basic understanding of Mg 2+ transport., (© 2022. The Author(s).)

RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function. This has been known for more than 50 years, and there are now thousands of in vitro studies, most of which have used ≥10 mM free Mg 2+ ions to achieve optimal RNA folding and function. In the cell, however, concentrations of free Mg 2+ ions are much lower, with most Mg 2+ ions chelated by metabolites. In this Perspective, we curate data from a number of sources to provide extensive summaries of cellular concentrations of metabolites that bind Mg 2+ and to estimate cellular concentrations of metabolite-chelated Mg 2+ species, in the representative prokaryotic and eukaryotic systems Escherichia coli , Saccharomyces cerevisiae , and iBMK cells. Recent research from our lab and others has uncovered the fact that such weakly chelated Mg 2+ ions can enhance RNA function, including its thermodynamic stability, chemical stability, and catalysis. We also discuss how metabolite-chelated Mg 2+ complexes may have played roles in the origins of life. It is clear from this analysis that bound Mg 2+ should not be simply considered non-RNA-interacting and that future RNA research, as well as protein research, could benefit from considering chelated magnesium.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca 2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca 2+ and Mg 2+ and a regulatory site (II) that has been thought to bind only Ca 2+ . Binding of Ca 2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca 2+ /Mg 2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg 2+ caused a 1.4-fold lower affinity for Ca 2+ . These experiments strongly suggest that cytosolic-free Mg 2+ occupies a significant population of the available site II. Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg 2+ , such as secondary hyperparathyroidism or ischemia, respectively., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg 2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg 2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg 2+ concentration. Subsequently, the second Mg 2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (K d  = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg 2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg 2+ concentrations. An increase of the Mg 2+ concentration from 0.25 to 10 mM increases nucleotide specificity (k cat /K m ) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg 2+ binds very weakly (K d ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg 2+ . Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases., Competing Interests: Conflict of interest K. A. J. is the President of KinTek Corp, which provided the AutoSF-120 stopped-flow, RQF-3 rapid-quench-flow, and KinTek Explorer software used in this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types, where they have an important role in Ca 2+ -dependent signalling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open in the physiological hyperpolarized resting membrane potential state (the so-called pump or P-state); thus, if not regulated, they would continuously leak Ca 2+ into cells. HACCs are permeable to Ca 2+ , Ba 2+ , and Mg 2+ ; activated by H 2 O 2 and the plant hormone abscisic acid (ABA); and their activity in guard cells is greatly reduced by increasing amounts of free cytosolic Ca 2+ ([Ca 2+ ] Cyt ), and hence closes during [Ca 2+ ] Cyt surges. Here, we demonstrate that the presence of the commonly used Mg-ATP inside the guard cell greatly reduces HACC activity, especially at voltages ≤ -200 mV, and that Mg 2+ causes this block. Therefore, we firstly conclude that physiological cytosolic Mg 2+ levels affect HACC gating and that channel opening requires either high negative voltages (≥ -200 mV) or displacement of Mg 2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with a Mg 2+ -sensitive animal inward-rectifying K + channel, we propose that the likely candidate HACCs described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved diacidic Mg 2+ binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg 2+ , much like in animal cells, is an important component in Ca 2+ signalling and homeostasis in plants.

Mechanical forces play a key role in essential biological processes including cell growth, division, deformation, adhesion, migration and intra-cell interactions. The effect of mechanical forces in modulating the structure and properties of metal-occupied protein binding sites has not been fully understood. Here, by employing a combination of density functional theory (DFT) calculations and polarizable continuum model (PCM) computations applied on model metal-loaded EF-hand binding sites, we shed light on the intimate mechanism of the Mg 2+ /Ca 2+ competition impacted by the application of mechanical stimuli. Applying mechanical force with a specific directionality and magnitude may shift the balance between the competing metal cations in favor of a given contestant depending on the composition and strength of the coordinative bonds and robustness of the metal binding site. Furthermore, the calculations help to determine the range of mechanical rupture forces typical for these structures: these range from 0.4 to 1.5 nN depending on the nature of the metal and amino acid residue. This positions the strength of the Mg 2+ -O and Ca 2+ -O coordinative bonds between that of typical covalent and hydrogen bonds. The bonds between the metal cation and the charged amino acid residue rupture at higher forces (∼1.2-1.5 nN) relative to those of their metal-noncharged counterparts which dissociate at ∼0.2-0.4 nN.

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg 2+ . We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg free diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg 2+ /Ca 2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.

P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca 2+ -ATP and Mg 2+ -ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development., Competing Interests: ML, YW, RB, FM, SS, MH No competing interests declared, JF, KS Reviewing editor, eLife

Protein phosphatase 5 (PP5), an important member of serine/threonine protein phosphatases, has been associated with diverse human cancers. Structural data demonstrate that two metal ions and two water molecules are located in the active site of PP5 and are involved in the dephosphorylations of the substrate. Recently, the catalytic reaction of PP5 has been studied by a density functional theory (DFT) method, and the role of the water molecules in regulating the substrate binding of PP5 was explored by our previous molecular dynamics (MD) study. These two studies enrich our understanding of the catalytic mechanism of PP5. However, the questions of how the metal ions regulate the substrate binding and the contribution of each metal ion to the catalysis remain not answered. To this end, the role of the catalytic Mn 2+ ions in regulating the binding of PP5 with its substrate was investigated through MD simulations. Results reveal that the different states of Mn 2+ ions can alter the structure of the active site and the conformations of the residues coordinating the Mn 2+ ions or the substrate. This leads to changes in the secondary structure and dynamical fluctuation of PP5, especially for that of helix αJ. With the position change of helix αJ, the volume of the substrate binding pocket is altered by the different states of Mn 2+ ions, indicating the regulatory role of Mn 2+ ions in PP5 activity. Furthermore, combined with the simulation data of our previous study, the sequential steps for PP5 binding of the substrate, the two active site water molecules, and the two Mn 2+ ions are proposed. This may help to develop new inhibitors to treat the PP5-related cancers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg2+, to catalyse the interconversion of the enantiomers of mandelate. Although the active site Mg2+ may be replaced by Mn2+, Co2+, or Ni2+, substitution by these metal ions does not markedly (<10-fold) alter the kinetic parameters Kappm, kappcat, and (kcat/Km)app for the substrates (R)- and (S)-mandelate, and the alternative substrate (S)-trifluorolactate. Viscosity variation experiments with Mn2+-MR showed that the metal ion plays a role in the uniform binding of the transition states for enzyme-substrate association, the chemical step, and enzyme-product dissociation. Surprisingly, the competitive inhibition constants (Ki) for inhibition of each metalloenzyme variant by benzohydroxamate did not vary significantly with the identity of the metal ion unlike the marked variation of the stability constants (K1) observed for M2+·BzH complex formation in solution. A similar trend was observed for the inhibition of the metalloenzyme variants by F-, except for Mg2+-MR, which bound F- tighter than would be predicted based on the stability constants for formation of M2+·F- complexes in solution. Thus, the enzyme modifies the enatic state of the bound metal ion cofactor so that the apparent electrophilicity of Mg2+ is enhanced, while that of Ni2+ is attenuated, resulting in a levelling effect relative to the trends observed for the free metals in solution.

The large yybP-ykoY family of bacterial riboswitches is broadly distributed phylogenetically. Previously, these gene-regulatory RNAs were proposed to respond to Mn 2+ . X-ray crystallography revealed a binuclear cation-binding pocket. This comprises one hexacoordinate site, with six oxygen ligands, which preorganizes the second, with five oxygen and one nitrogen ligands. The relatively soft nitrogen ligand was proposed to confer affinity for Mn 2+ , but how this excludes other soft cations remained enigmatic. By subjecting representative yybP-ykoY riboswitches to diverse cations in vitro, we now find that these RNAs exhibit limited transition metal ion selectivity. Among the cations tested, Cd 2+ and Mn 2+ bind most tightly, and comparison of three new Cd 2+ -bound crystal structures suggests that these riboswitches achieve selectivity by enforcing heptacoordination (favored by high-spin Cd 2+ and Mn 2+ , but otherwise uncommon) in the softer site. Remarkably, the Cd 2+ - and Mn 2+ -selective bacterial transcription factor MntR also uses heptacoordination within a binuclear site to achieve selectivity., (Published by Elsevier Ltd.)

Memantine and ketamine are NMDA receptor (NMDAR) open channel blockers that are thought to act via similar mechanisms at NMDARs, but exhibit divergent clinical effects. Both drugs act by entering open NMDARs and binding at a site deep within the ion channel (the deep site) at which the endogenous NMDAR channel blocker Mg 2+ also binds. Under physiological conditions, Mg 2+ increases the IC 50 s of memantine and ketamine through competition for binding at the deep site. Memantine also can inhibit NMDARs after associating with a second site accessible in the absence of agonist, a process termed second site inhibition (SSI) that is not observed with ketamine. Here we investigated the effects of 1 mM Mg 2+ on recovery from inhibition by memantine and ketamine, and on memantine SSI, of the four main diheteromeric NMDAR subtypes. We found that: recovery from memantine inhibition depended strongly on the concentration of memantine used to inhibit the NMDAR response; Mg 2+ accelerated recovery from memantine and ketamine inhibition through distinct mechanisms and in an NMDAR subtype-dependent manner; and Mg 2+ occupation of the deep site disrupted memantine SSI in a subtype-dependent manner. Our results support the hypothesis that memantine associates with, but does not inhibit at the second site. After associating with the second site, memantine can either slowly dissociate directly to the extracellular solution, or transit to the deep site, resulting in typical channel block. Memantine's relatively slow dissociation from the second site underlies the dependence of NMDAR recovery from inhibition on both memantine concentration and on Mg 2+ ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Magnesium ions (Mg 2+ ) are divalent cations essential for various cellular functions. Mg 2+ homeostasis is maintained through Mg 2+ channels such as MgtE, a prokaryotic Mg 2+ channel whose gating is regulated by intracellular Mg 2+ levels. Our previous crystal structure of MgtE in the Mg 2+ -bound, closed state revealed the existence of seven crystallographically-independent Mg 2+ -binding sites, Mg1-Mg7. The role of Mg 2+ -binding to each site in channel closure remains unknown. Here, we investigated Mg 2+ -dependent changes in the structure and dynamics of MgtE using nuclear magnetic resonance spectroscopy. Mg 2+ -titration experiments, using wild-type and mutant forms of MgtE, revealed that the Mg 2+ binding sites Mg1, Mg2, Mg3, and Mg6, exhibited cooperativity and a higher affinity for Mg 2+ , enabling the remaining Mg 2+ binding sites, Mg4, Mg5, and Mg7, to play important roles in channel closure. This study revealed the role of each Mg 2+ -binding site in MgtE gating, underlying the mechanism of cellular Mg 2+ homeostasis., Competing Interests: TM, SI, TK, MH, RI, ON, KI, AM, IS, MO No competing interests declared, (© 2018, Maruyama et al.)

Though the structure of the substrate stem loop I (SLI)-stem loop V (SLV) kissing loop junction of the Varkud Satellite ribozyme has been experimentally characterized, the dynamics of this Mg 2+ -dependent loop-loop interaction have been elusive. Specifically, each hairpin loop contains a U-turn motif, but only SLV shows a conformational shift triggered by Mg 2+ ion association. Here, we use molecular dynamics simulations to analyze the binding and dynamics of this kissing loop junction. We show that SLV acts as a scaffold, providing stability to the junction. Mg 2+ ions associate with SLV when it is part of the junction in a manner similar to when it is unbound, but there is no specificity in Mg 2+ binding for the SLI loop. This suggests that the entropic penalty of ordering the larger SLI is too high, allowing SLV to act as a scaffold for multiple substrate loop sequences., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The S-adenosylmethionine (SAM)-I riboswitch is a noncoding RNA that regulates the transcription termination process in response to metabolite (SAM) binding. The aptamer portion of the riboswitch may adopt an open or closed state depending on the presence of metabolite. Although the transition between the open and closed states is critical for the switching process, its atomistic details are not well understood. Using atomistic simulations, we calculate the effect of SAM and magnesium ions on the folding free energy landscape of the SAM-I riboswitch. These molecular simulation results are consistent with our previous wetlab experiments and aid in interpreting the SHAPE probing measurements. Here, molecular dynamics simulations explicitly identify target RNA motifs sensitive to magnesium ions and SAM. In the simulations, we observe that, whereas the metabolite mostly stabilizes the P1 and P3 helices, magnesium serves an important role in stabilizing a pseudoknot interaction between the P2 and P4 helices, even at high metabolite concentrations. The pseudoknot stabilization by magnesium, in combination with P1 stabilization by SAM, explains the requirement of both SAM and magnesium to form the fully collapsed metabolite-bound closed state of the SAM-I riboswitch. In the absence of SAM, frequent open-to-closed conformational transitions of the pseudoknot occur, akin to breathing. These pseudoknot fluctuations disrupt the binding site by facilitating fluctuations in the 5'-end of helix P1. Magnesium biases the landscape toward a collapsed state (preorganization) by coordinating pseudoknot and 5'-P1 fluctuations. The cooperation between SAM and magnesium in stabilizing important tertiary interactions elucidates their functional significance in transcription regulation., (Published by Elsevier Inc.)

Reverse Watson-Crick G:C basepairs (G:C W:W Trans) occur frequently in different functional RNAs. This is one of the few basepairs whose gas-phase-optimized isolated geometry is inconsistent with the corresponding experimental geometry. Several earlier studies indicate that through post-transcriptional modification, direct protonation, or coordination with Mg 2+ , accumulation of positive charge near N7 of guanine can stabilize the experimental geometry. Interestingly, recent studies reveal significant variation in the position of putatively bound Mg 2+ . This, in conjunction with recently raised doubts regarding some of the Mg 2+ assignments near the imino nitrogen of guanine, is suggestive of the existence of multiple Mg 2+ binding modes for this basepair. Our detailed investigation of Mg 2+ -bound G:C W:W Trans pairs occurring in high-resolution RNA crystal structures shows that they are found in 14 different contexts, eight of which display Mg 2+ binding at the Hoogsteen edge of guanine. Further examination of occurrences in these eight contexts led to the characterization of three different Mg 2+ binding modes: 1) direct binding via N7 coordination, 2) direct binding via O6 coordination, and 3) binding via hydrogen-bonding interaction with the first-shell water molecules. In the crystal structures, the latter two modes are associated with a buckled and propeller-twisted geometry of the basepair. Interestingly, respective optimized geometries of these different Mg 2+ binding modes (optimized using six different DFT functionals) are consistent with their corresponding experimental geometries. Subsequent interaction energy calculations at the MP2 level, and decomposition of its components, suggest that for G:C W:W Trans , Mg 2+ binding can fine tune the basepair geometries without compromising with their stability. Our results, therefore, underline the importance of the mode of binding of Mg 2+ ions in shaping RNA structure, folding and function., (Copyright © 2017. Published by Elsevier Inc.)

N -methyl-d-aspartate receptors (NMDARs) govern synaptic plasticity, development, and neuronal response to insult. Prolonged activation of NMDARs such as during an insult may activate secondary currents or modulate Mg 2+ sensitivity, but the conditions under which these occur are not fully defined. We reexamined the effect of prolonged NMDAR activation in juvenile mouse hippocampal slices. NMDA (10 μM) elicited current with the expected negative-slope conductance in the presence of 1.2 mM Mg 2+ However, several minutes of continued NMDA exposure elicited additional inward current at -70 mV. A higher concentration of NMDA (100 µM) elicited the current more rapidly. The additional current was not dependent on Ca 2+ , network activity, or metabotropic NMDAR function and did not persist on agonist removal. Voltage ramps revealed no alteration of either reversal potential or NMDA-elicited conductance between -30 mV and +50 mV. The result was a more linear NMDA current-voltage relationship. The current linearization was also induced in interneurons and in mature dentate granule neurons but not immature dentate granule cells, dissociated cultured hippocampal neurons, or nucleated patches excised from CA1 pyramidal neurons. Comparative simulations of NMDA application to a CA1 pyramidal neuron and to a cultured neuron revealed that linearization can be explained by space-clamp errors arising from gradual recruitment of distal dendritic NMDARs. We conclude that persistent secondary currents do not strongly contribute to NMDAR responses in juvenile mouse hippocampus and careful discernment is needed to exclude contributions of clamp artifacts to apparent secondary currents. NEW & NOTEWORTHY We report that upon sustained activation of NMDARs in juvenile mouse hippocampal neurons there is apparent loss of Mg 2+ block at negative membrane potentials. However, the phenomenon is explained by loss of dendritic voltage clamp, leading to a linear current-voltage relationship. Our results give a specific example of how spatial voltage errors in voltage-clamp recordings can readily be misinterpreted as biological modulation., (Copyright © 2017 the American Physiological Society.)

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn 2+ -dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn 2+ , rather than Mg 2+ , as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn 2+ and Mg 2+ , respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn 2+ than with Mg 2+ . Notably, the substitution fidelity of PrimPol in the presence of Mn 2+ was determined to be in the range of 3.4×10 -2 to 3.8×10 -1 , indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn 2+ and 150-4500 with Mg 2+ , and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Calcium (Ca(2+)) flux into the matrix is tightly controlled by the mitochondrial Ca(2+) uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca(2+) or Mg(2+) destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg(2+) loading or blockade of mitochondrial Ca(2+) extrusion suppresses MCU Ca(2+)-uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca(2+) binding proteins.

Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.

Previous molecular dynamics (MD) simulations of ion-lipid interactions have focused on pure phospholipid bilayers. Many functional microdomains in membranes have a complex composition of cholesterol and phospholipids. Here, we reveal the distinctiveness of the interactions and the effects of the ions on a cholesterol-rich bilayer by performing MD simulations of a cholesterol-rich bilayer with a Na(+)/K(+) mixture or a Na(+)/K(+)/Ca(2+)/Mg(2+) mixture. The simulations reveal that Ca(2+) maintains its dominant role in the interaction with the cholesterol-rich bilayer, but the binding affinity of Mg(2+) to the cholesterol-rich bilayer is even weaker than the affinities of Na(+) and K(+), whereas its interaction with pure phospholipid bilayers is strong and is only slightly weaker than that of Ca(2+). Additionally, it was found that the presence of additional divalent cations induces the headgroups of phospholipids to be more perpendicular to the membrane surface, reducing the lateral movement of lipids and slightly altering the ordering and packing of the cholesterol-rich bilayer, different from divalent cations, which strongly influence that ordering and packing of pure phospholipid bilayers. Therefore, this study indicates that cholesterol in the membrane could affect the interactions between membrane and cations. The findings could be helpful in understanding the biological processes relevant to regulation of cations in cholesterol-rich regions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Based on melamine binding-triggered triplex formation and subsequent activation of Mg(2+)-dependent DNAzymes, a novel strategy for DNAzyme regulation was proposed and developed for melamine recognition.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Magnesium (Mg) is the second most abundant cation in living cells. Over 300 enzymes are known to be Mg-dependent, and changes in the Mg concentration significantly affects the membrane potential. As Mg becomes deficient, starch accumulation and chlorosis, bridged by the generation of reactive oxygen species, are commonly found in Mg-deficient young mature leaves. These defects further cause the inhibition of photosynthesis and finally decrease the biomass. Recently, transcriptome analysis has indicated the transcriptinal downregulation of chlorophyll apparatus at the earlier stages of Mg deficiency, and also the potential involvement of complicated networks relating to hormonal signaling and circadian oscillation. However, the processes of the common symptoms as well as the networks between Mg deficiency and signaling are not yet fully understood. Here, for the purpose of defining the missing pieces, several problems are considered and explained by providing an introduction to recent reports on physiological and transcriptional responses to Mg deficiency. In addition, it has long been unclear whether the Mg deficiency response involves the modulation of Mg2+ transport system. In this review, the current status of research on Mg2+ transport and the relating transporters are also summarized. Especially, the rapid progress in physiological characterization of the plant MRS2 gene family as well as the fundamental investigation about the molecular mechanism of the action of bacterial CorA proteins are described.

Background: The influence of extracellular calcium on phagocytosis is limited, and the involvement of extracellular magnesium is unclear., Aims: The role of extracellular calcium and magnesium on phagocytosis efficiency was investigated., Results: Extracellular calcium had no influence on the internalization of 1 μm polystyrene particles by primary monocytes as has been shown before for the human lymphoma-derived, differentiated cell line U937 and murine alveolar macrophage-derived MH-S cells. In contrast, the phagocytosis by differentiated U937 cells was positively affected by the presence of extracellular magnesium whereas that of MH-S cells was not. An extracellular increase in the magnesium level did not cause an increase in the free cytosolic magnesium concentration in either cell line. In contrast to magnesium, extracellular calcium caused an increase in intracellular divalent cation levels in differentiated U937 cells., Conclusions: A phagocytosis-enhancing effect in the extracellular space was observed in relation to extracellular magnesium rather than with an intracellular increase in magnesium levels, indicating that murine and human immune cells might be regulated differently.

In this study, we investigated the effect of different multivalent cations on granule formation. Previous experiments showed that formation of matrix EPS and their structure depend of the presence of divalent cations. This study indicates that trivalent cations are also playing an important role. However, the more compact granules were formed in the presence of all cations. The authors tried also to determine changes in proteomic profile of slime and tightly bound EPS. These results showed that matrix EPS is composed of a variety of large and complex proteins, but there are also small proteins, like for example, lectins. These small proteins have a role in the interaction between cells and exopolysaccharides and in granules formation.

Unlabelled: The fidelity of human immunodeficiency virus (HIV) reverse transcriptase (RT) has been a subject of intensive investigation. The mutation frequencies for the purified enzyme in vitro vary widely but are typically in the 10(-4) range (per nucleotide addition), making the enzyme severalfold less accurate than most polymerases, including other RTs. This has often been cited as a factor in HIV's accelerated generation of genetic diversity. However, cellular experiments suggest that HIV does not have significantly lower fidelity than other retroviruses and shows a mutation frequency in the 10(-5) range. In this report, we reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results from experiments conducted with physiological (for lymphocytes) concentrations of free Mg(2+) (~0.25 mM) and is comparable to Moloney murine leukemia virus (MuLV) RT fidelity. The physiological conditions produced mutation rates that were 5 to 10 times lower than those obtained under typically employed in vitro conditions optimized for RT activity (5 to 10 mM Mg(2+)). These results were consistent in both commonly used lacZα complementation and steady-state fidelity assays. Interestingly, although HIV RT showed severalfold-lower fidelity under high-Mg(2+) (6 mM) conditions, MuLV RT fidelity was insensitive to Mg(2+). Overall, the results indicate that the fidelity of HIV replication in cells is compatible with findings of experiments carried out in vitro with purified HIV RT, providing more physiological conditions are used., Importance: Human immunodeficiency virus rapidly evolves through the generation and subsequent selection of mutants that can circumvent the immune response and escape drug therapy. This process is fueled, in part, by the presumably highly error-prone HIV polymerase reverse transcriptase (RT). Paradoxically, results of studies examining HIV replication in cells indicate an error frequency that is ~10 times lower than the rate for RT in the test tube, which invokes the possibility of factors that make RT more accurate in cells. This study brings the cellular and test tube results in closer agreement by showing that HIV RT is not more error prone than other RTs and, when assayed under physiological magnesium conditions, has a much lower error rate than in typical assays conducted using conditions optimized for enzyme activity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Lysosomal Ca(2+) homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca(2+) signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg(2+) and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg(2+) specifically inhibited TPC2 outward current, whereas lysosomal Mg(2+) partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg(2+), TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca(2+) release in intact cells is regulated by Mg(2+), PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca(2+) signaling and link this pathway to Mg(2+) homeostasis and MAP kinases, pointing to roles for lysosomal Ca(2+) in cell growth, inflammation and cancer.

Metal cations represent key elements of RNA structure and function. In the Neurospora VS ribozyme, metal cations play diverse roles; they are important for substrate recognition, formation of the active site, and shifting the pKa's of two key nucleobases that contribute to the general acid-base mechanism. Recently, we determined the NMR structure of the A730 loop of the VS ribozyme active site (SLVI) that contributes the general acid (A756) in the enzymatic mechanism of the cleavage reaction. Our studies showed that magnesium (Mg(2+)) ions are essential to stabilize the formation of the S-turn motif within the A730 loop that exposes the A756 nucleobase for catalysis. In this article, we extend these NMR investigations by precisely mapping the Mg(2+)-ion binding sites using manganese-induced paramagnetic relaxation enhancement and cadmium-induced chemical-shift perturbation of phosphorothioate RNAs. These experiments identify five Mg(2+)-ion binding sites within SLVI. Four Mg(2+) ions in SLVI are associated with known RNA structural motifs, including the G-U wobble pair and the GNRA tetraloop, and our studies reveal novel insights about Mg(2+) ion binding to these RNA motifs. Interestingly, one Mg(2+) ion is specifically associated with the S-turn motif, confirming its structural role in the folding of the A730 loop. This Mg(2+) ion is likely important for formation of the active site and may play an indirect role in catalysis.

The TRP gene family encodes primarily cation non-selective, Ca2+ permeant channels that are involved in a dizzying array of sensory mechanisms. Two channels in this large family TRPV5 and TRPV6 are highly Ca2+ selective and are expressed in epithelia where they are important in Ca2+ uptake. TRPV5/6 are constitutively active, yet the mechanisms regulating their activation in native tissue remains elusive. Here we functionally characterize the Xenopus TRPV6 homolog. xTRPV6 is expressed in the oocyte and encodes a channel that is permeant to divalents including Ca2+ , and displays a high permeability to Mg2+ . The oocyte does not exhibit functional TRPV6-like current at rest, showing that the endogenous channel is somehow maintained in an inactive state. We show that endogenous as well as overexpressed xTRPV6 interacts with xTRPC1 and that this interaction inhibits xTRPV6 currents. As such TRPC1 is likely to regulate the activity of TRPV6 under physiological conditions., (Copyright © 2013 Wiley Periodicals, Inc.)

Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5'-deoxyribose phosphate and the 3'-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3'-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm3+ or Pb2+ in the active site. However, these are poor surrogates for Mg2+ because Sm3+ is not a cofactor and Pb2+ inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg2+ ion in the active site. The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules. One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme-product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.

Background: Dietzia natronolimnaea is one of the most important bacterial bioresources for high efficiency canthaxanthin production. It produces the robust and stable pigment canthaxanthin, which is of special interest for the development of integrated biorefineries. Mutagenesis employing 12C6+ irradiation is a novel technique commonly used to improve microorganism productivity. This study presents a promising route to obtaining the highest feasible levels of biomass dry weight (BDW), and total canthaxanthin by using a microdosimetric model of 12C6+ irradiation mutation in combination with the optimization of nutrient medium components., Results: This work characterized the rate of both lethal and non-lethal dose mutations for 12C6+ irradiation and the microdosimetric kinetic model using the model organism, D. natronolimnaea svgcc1.2736. Irradiation with 12C6+ ions resulted in enhanced production of canthaxanthin, and is therefore an effective method for strain improvement of D. natronolimnaea svgcc1.2736. Based on these results an optimal dose of 0.5-4.5 Gy, Linear energy transfer (LET) of 80 keV μm-1and energy of 60 MeV u-1 for 12C6+ irradiation are ideal for optimum and specific production of canthaxanthin in the bacterium. Second-order empirical calculations displaying high R-squared (0.996) values between the responses and independent variables were derived from validation experiments using response surface methodology. The highest canthaxanthin yield (8.14 mg) was obtained with an optimized growth medium containing 21.5 g L-1 D-glucose, 23.5 g L-1 mannose and 25 ppm Mg2+ in 1 L with an irradiation dose of 4.5 Gy., Conclusions: The microdosimetric 12C6+ irradiation model was an effective mutagenic technique for the strain improvement of D. natronolimnaea svgcc1.2736 specifically for enhanced canthaxanthin production. At the very least, random mutagenesis methods using 12C6+ions can be used as a first step in a combined approach with long-term continuous fermentation processes. Central composite design-response surface methodologies (CCD-RSM) were carried out to optimize the conditions for canthaxanthin yield. It was discovered D-glucose, Mg2+ and mannose have significant influence on canthaxanthin biosynthesis and growth of the mutant strain.

The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg (2+) and Mn (2+) but not Ca (2+) and Zn (2+). Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.

Gap junction (GJ) channels composed of Connexin36 (Cx36) are widely expressed in the mammalian CNS and form electrical synapses between neurons. Here we describe a novel modulatory mechanism of Cx36 GJ channels dependent on intracellular free magnesium ([Mg(2+)]i). We examined junctional conductance (gj) and its dependence on transjunctional voltage (Vj) at different [Mg(2+)]i in cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg(2+)]i. Thus, gj can be augmented or reduced by lowering or increasing [Mg(2+)]i, respectively. Similar changes in gj and Vj-gating were observed using MgATP or K2ATP in pipette solutions, which increases or decreases [Mg(2+)]i, respectively. Changes in phosphorylation of Cx36 or in intracellular free calcium concentration were not involved in the observed Mg(2+)-dependent modulation of gj. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg(2+)]i resulted in asymmetric Vj-gating. The gj of GJs formed of Cx26, Cx32, Cx43, Cx45, and Cx47 was also reduced by increasing [Mg(2+)]i, but was not increased by lowering [Mg(2+)]i; single-channel conductance did not change. We showed that [Mg(2+)]i affects both open probability and the number of functional channels, likely through binding in the channel lumen. Finally, we showed that Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus in rat brain slices are similarly affected by changes in [Mg(2+)]i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg(2+)]i, are modified.

Aldehyde dehydrogenase 1 (ALDH1A1) catalyzes the oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) ions decrease ALDH1 activity in part by increasing NADH binding affinity to the enzyme. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH in solution (τ=0.4 ns) and two enzyme-bound NADH states (τ=2.0 ns and τ=7.7 ns). We used this technique to investigate the effects of Mg(2+) ions on the ALDH1A1-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD values for both NADH conformations in ALDH1A1 ranged from about 24 μM to 1 μM for Mg(2+) ion concentrations of 0-6000 μM, respectively. The rate constants for dissociation of the enzyme-NADH complex ranged from 0.03 s(-1) (6000 μM Mg(2+)) to 0.30s(-1) (0 μM Mg(2+)) as determined by addition of excess NAD(+) to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. During the initial reaction of enzyme with NAD(+) and butyraldehyde, there was an immediate rise in the NADH fluorescence, due to the formation of bound NADH complexes, with a constant steady-state rate of production of free NADH. As the Mg(2+) ion concentration was increased, there was a consistent decrease of the enzyme catalytic turnover from 0.31 s(-1) (0 μM Mg(2+)) to 0.050 s(-1) (6000 μM Mg(2+)) and a distinct shift in steady-state conformational population from one that favors the ALDH1-NADH complex with the shorter fluorescence lifetime (33% excess) in the absence of magnesium ion to one that favors the ALDH1-NADH complex with the longer fluorescence lifetime (13% excess) at 6000 μM Mg(2+). This shift in conformational population at higher Mg(2+) ion concentrations and to lower enzyme activity may be due to longer residence time of the NADH in the ALDH1 pocket. The results from monitoring enzyme catalysis in the absence of magnesium suggests that the ALDH1-NADH complex with the shorter fluorescence lifetime is the form initially produced, and the complex with the longer fluorescence lifetime is produced through isomerization., (Published by Elsevier Ireland Ltd.)

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1)., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Polcalcins are small EF-hand proteins believed to assist in regulating pollen-tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain-swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg(2+), or 100 μM Ca(2+). Phl p 7 is monomeric in all three ligation states. In the apo-form, both EF-hand motifs reside in the closed conformation, with roughly antiparallel N- and C-terminal helical segments. In 5.0 mM Mg(2+), the divalent ion is bound by EF-hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca(2+)-bound Phl p 7 resembles that previously reported for Bet v 4-likewise exposing apolar surface to the solvent. Occluded in the apo- and Mg(2+)-bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF-hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at -x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca(2+). When Mg(2+) resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer-sphere ligand. (15)N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states., (Copyright © 2012 Wiley Periodicals, Inc.)

Intracellular Ca(2+)-binding proteins (CaBPs) are sensors of the calcium signal and several of them even shape the signal. Most of them are equipped with at least two EF-hand motifs designed to bind Ca(2+). Their affinities are very variable, can display cooperative effects, and can be modulated by physiological Mg(2+) concentrations. These binding phenomena are monitored by four major techniques: equilibrium dialysis, fluorimetry with fluorescent Ca(2+) indicators, flow dialysis, and isothermal titration calorimetry. In the last quarter of the twentieth century reports on the ion-binding characteristics of several abundant wild-type CaBPs were published. With the advent of recombinant CaBPs it became possible to determine these properties on previously inaccessible proteins. Here I report on studies by our group carried out in the last decade on eight families of recombinant CaBPs, their mutants, or truncated domains. Moreover this chapter deals with the currently used methods for quantifying the binding of Ca(2+) and Mg(2+) to CaBPs.

Sodium-calcium exchange across plasma membrane is regulated by intracellular calcium ions. The sodium-calcium exchanger (NCX1) is activated by successive saturation of numerous Ca(2+)-binding sites located in the intracellular loop of the protein. The progressive saturation of the binding domain CBD12 by Ca(2+) results in a series of conformational changes of CBD12 as well as of entire NCX1 molecule. Like other soluble and membrane Ca(2+)-binding proteins, NCX1 can also be regulated by Mg(2+) that antagonises Ca(2+) at the level of divalent cation-binding sites. This chapter summarises data on Mg(2+) impacts in the cells. Regulatory action of Mg(2+) on intracellular Ca(2+)-dependent processes can be achieved due to changes of its cytoplasmic level, which take place in the range of [Mg(2+)](i) from 0.5 to 3 mM. Under normal conditions, these changes are ensured by activation of plasmalemmal Mg(2+) transport systems and by variations in ATP level in cytoplasm. In heart and in brain, some pathological conditions, such as hypoxia, ischemia and ischemia followed by reperfusion, are associated with an important increase in intracellular Ca(2+). The tissue damage due to Ca(2+) overload may be prevented by Mg(2+). The protective actions of Mg(2+) can be achieved due to its ability to compete with Ca(2+) for the binding sites in a number of proteins responsible for the rise in intracellular free Ca(2+), including NCX1, in case when the reverse mode of Na(+)/Ca(2+) exchange becomes predominant. Saturation of CBD12 by Mg(2+) results in important changes of NCX1 conformation. Modulating actions of Mg(2+) on the conformation of NCX1 were detected at a narrow range of Mg(2+) concentration, from 0.5 to 1 mM. These data support an idea that variations of intracellular Mg(2+) could modify transmembrane Ca(2+) movements ensured by NCX1.

Cytoskeletal reorganization processes can be analyzed by studying the nanometer-scale spontaneous motion of beads bound to the cytoskeleton. The bead motion is determined by force fluctuations within the cytoskeletal network that originates from myosin motor activity and dynamic restructuring of cytoskeletal filaments. We investigated to what extend the spontaneous bead motion is influenced by the dynamics of the link between the bead and the cytoskeleton in the presence of divalent cations. Our data show that, when K562 cells expressing constitutively (alpha 5 beta 1) integrin and when stably transfected with (alpha v beta 3) integrin, spontaneous bead motion is dramatically affected by the presence of 1mM Mn(2+) (integrin, activate state) compared to 1mM Ca(2+)/Mg(2+) ions (integrin, inactive state). The directionality of the bead motion, which is influenced by the overall stability of the cytoskeletal network and by actomyosin-generated forces, is markedly different, whilst the persistence remained similar due to the specific binding of either Mn(2+) or Mg(2+)/Ca(2+) ions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) ≫ Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.

Mg(2+)-dependent ATPases were investigated in Malpighian tubules of the blood-sucking insect, Triatoma infestans, with cytochemical procedures for light and electron microscopy. The aim was to establish patterns of enzyme occurrence in the blood-sucking insect under control rearing conditions for further comparisons with animals subjected to the action of stress factors. Enzyme activity was found in laminated "concretions" present in distal cells, in edges of urate crystals at the lumen of the proximal region of tubules, in the basement membrane of proximal cells, and variously distributed in plasmalemma invaginations of both distal and proximal cells. Presence of ATPases in the "concretions" and urate crystals is presumed to be due to engulfment of other ATPase-containing components during formation of these structures. Cytochemical reactivity in the basement membrane and plasmalemma invaginations is assumed to be involved with active transport of waste molecules from and to hemolymph and differs as a function of the Malpighian tubule region. This paper provides a basic understanding of the enzyme occurrence in the blood sucking insects, and can be used as a pattern for comparative means of the staining patterns among Triatominae species., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Eukaryotic translation termination is governed by eRF1 and eRF3. eRF1 recognizes the stop codons and then hydrolyzes peptidyl-tRNA. eRF3, which facilitates the termination process, belongs to the GTPase superfamily. In this study, the effect of the MC domain of eRF1a (eRF1aMC) on the GTPase activity of eRF3 was analyzed using fluorescence spectra and high-performance liquid chromatography. The results indicated eRF1aMC promotes the GTPase activity of eRF3, which is similar to the role of eRF1a. Furthermore, the increased affinity of eRF3 for GTP induced by eRF1aMC was dependent on the concentration of Mg(2+). Changes in the secondary structure of eRF3C after binding GTP/GDP were detected by CD spectroscopy. The results revealed changes of conformation during formation of the eRF3C·GTP complex that were detected in the presence of eRF1a or eRF1aMC. The conformations of the eRF3C·eRF1a·GTP and eRF3C·eRF1aMC·GTP complexes were further altered upon the addition of Mg(2+). By contrast, there was no change in the conformation of GTP bound to free eRF3C or the eRF3C·eRF1aN complex. These results suggest that alterations in the conformation of GTP bound to eRF3 is dependent on eRF1a and Mg(2+), whereas the MC domain of eRF1a is responsible for the change in the conformation of GTP bound to eRF3 in Euplotes octocarinatus.

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.