Competitive Binding of Mg2+ and Na+ Ions to Nucleic Acids: From Helices to Tertiary Structures

Nucleic acids generally reside in cellular aqueous solutions with mixed divalent/monovalent ions, and the competitive binding of divalent and monovalent ions is critical to the structures of nucleic acids because of their polyanionic nature. In this work, we first proposed a general and effective method for simulating a nucleic acid in mixed divalent/monovalent ion solutions with desired bulk ion concentrations via molecular dynamics (MD) simulations and investigated the competitive binding of Mg²⁺/Na⁺ ions to various nucleic acids by all-atom MD simulations. The extensive MD-based examinations show that single MD simulations conducted using the proposed method can yield desired bulk divalent/monovalent ion concentrations for various nucleic acids, including RNA tertiary structures. Our comprehensive analyses show that the global binding of Mg²⁺/Na⁺ to a nucleic acid is mainly dependent on its structure compactness, as well as Mg²⁺/Na⁺ concentrations, rather than the specific structure of the nucleic acid. Specifically, the relative global binding of Mg²⁺ over Na⁺ is stronger for a nucleic acid with higher effective surface charge density and higher relative Mg²⁺/Na⁺ concentrations. Furthermore, the local binding of Mg²⁺/Na⁺ to a phosphate of a nucleic acid mainly depends on the local phosphate density in addition to Mg²⁺/Na⁺ concentrations.     

Solution conformation of various uridine diphosphoglucose salts as probed by NMR spectroscopy

The solution conformations of uridine diphosphoglucose (UDP-Glc) under a variety of conditions (solvent, ionic strength, various mono- and divalent cations) have been studied by NMR spectroscopy (1H, 13C, 31P, and 25Mg). In the case of divalent cations (Ca2+, Mg2+, Mn2+) the phosphate oxygens are the preferred coordination sites and analysis of the 25Mg linewidths of solutions with various [Mg2+]/[UDP-Glc] ratios, indicates that the 1:1 Mg2+ UDP-Glc complex is the major species. From 13C relaxation data and hydrodynamic theory, it has been demonstrated that under all conditions UDP-Glc adopts a fairly extended overall shape and that magnesium ions lead to a significant increase in the average length of the UDP-Glc molecule as compared to monovalent cations. Thus, one of the roles of the metal ion in enzymic reactions involving nucleotide sugars may be to preorganize the nucleotide sugar.     

Monovalent ion stimulated adenosine triphosphatase from oat roots

Monovalent ion stimulated ATPase activity from oat (Avena sativa) roots has been found to be associated with various membrane fractions (cell wall, mitochondrial and microsomal) of oat roots. The ATPase requires Mg²⁺ (or Mn⁺²) but is further stimulated by K⁺ and other monovalent ions. The monovalent ions are ineffective in the absence of the divalent activating cation. The ATPase has been described with respect to monovalent ion specificity, temperature, pH, substrate specificity, and Mg²⁺ and K⁺ concentrations. It was further shown that oligomycin inhibits a part of the total ATPase activity and on the basis of the oligomycin sensitivity it appears that at least 2 membrane associated ATPases are being measured. The mitochondrial fraction is most sensitive to oligomycin and the microsomal fraction is least sensitive to oligomycin. The oligomycin insensitive ATPase appears to be stimulated more by K⁺ than the oligomycin sensitive ATPase.     

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K⁺ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg⁺² is optimal for the physiological reaction, however, Co⁺², Mn⁺², and Ni⁺² also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.     

Mechanism of μ-Opioid Receptor-Magnesium Interaction and Positive Allosteric Modulation

In the era of opioid abuse epidemics, there is an increased demand for understanding how opioid receptors can be allosterically modulated to guide the development of more effective and safer opioid therapies. Among the modulators of the μ-opioid (MOP) receptor, which is the pharmacological target for the majority of clinically used opioid drugs, are monovalent and divalent cations. Specifically, the monovalent sodium cation (Na⁺) has been known for decades to affect MOP receptor signaling by reducing agonist binding, whereas the divalent magnesium cation (Mg²⁺) has been shown to have the opposite effect, notwithstanding the presence of sodium chloride. Although ultra-high-resolution opioid receptor crystal structures have revealed a specific Na⁺ binding site and molecular dynamics (MD) simulation studies have supported the idea that this monovalent ion reduces agonist binding by stabilizing the receptor inactive state, the putative binding site of Mg²⁺ on the MOP receptor, as well as the molecular determinants responsible for its positive allosteric modulation of the receptor, are unknown. In this work, we carried out tens of microseconds of all-atom MD simulations to investigate the simultaneous binding of Mg²⁺ and Na⁺ cations to inactive and active crystal structures of the MOP receptor embedded in an explicit lipid-water environment and confirmed adequate sampling of Mg²⁺ ion binding with a grand canonical Monte Carlo MD method. Analyses of these simulations shed light on 1) the preferred binding sites of Mg²⁺ on the MOP receptor, 2) details of the competition between Mg²⁺ and Na⁺ cations for specific sites, 3) estimates of binding affinities, and 4) testable hypotheses of the molecular mechanism underlying the positive allosteric modulation of the MOP receptor by the Mg²⁺ cation.     

88 Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamic simulation and Poisson–Boltzmann approach

Ion interactions with nucleic acids (both DNA and RNA) are an important and evolving field of investigation. Positively charged cations may interact with highly negatively charged nucleic acids via simple electrostatic interactions to help screen the electrostatic repulsion along the nucleic acids and assist their folding and/or compaction. Cations may also bind at specific sites and become integral parts of the structures, possibly playing important enzymatic roles. Two popular methods for computationally exploring a nucleic acid’s ion atmosphere are atomistic molecular dynamics (MD) simulations and the Poisson–Boltzmann (PB) equation. In general, monovalent ion results obtained from MD simulations and the PB equation agree well with experiment. However, Bai et al. (2007) observed discrepancies between experiment and the PB equation while examining the competitive binding of monovalent and divalent ions, with more significant discrepancies for divalent ions. The goal of this project was to thoroughly investigate monovalent (Na⁺) and divalent (Mg²⁺) ion distributions formed around a DNA duplex with MD simulations and the PB equation. We simulated three different cation concentrations, and matched the equilibrated bulk ion concentration for our theoretical calculations with the PB equation. Based on previous work, our Mg²⁺ ions were fully solvated, the expected state of Mg²⁺ ions when interacting with a duplex, when the production simulations began and remained throughout the simulations (Kirmizialtin, 2010; Robbins, 2012). Na⁺ ion distributions and number of Na⁺ ions within 10?Å of the DNA obtained from our two methods agreed well. However, results differed for Mg²⁺ ions, with a lower number of ions within the cut-off distance obtained from the PB equation when compared to MD simulations. The Mg²⁺ ion distributions around the DNA obtained via the two methods also differed. Based on our results, we conclude that the PB equation will systematically underestimate Mg²⁺ ions bound to DNA, and much of this deviation is attributed to dielectric saturation associated with high valency ions.     

Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamics simulation and a Poisson‐Boltzmann approach

The ion atmosphere created by monovalent (Na⁺) or divalent (Mg²⁺) cations surrounding a B‐form DNA duplex were examined using atomistic molecular dynamics (MD) simulations and the nonlinear Poisson‐Boltzmann (PB) equation. The ion distributions predicted by the two methods were compared using plots of radial and two‐dimensional cation concentrations and by calculating the total number of cations and net solution charge surrounding the DNA. Na⁺ ion distributions near the DNA were more diffuse in PB calculations than in corresponding MD simulations, with PB calculations predicting lower concentrations near DNA groove sites and phosphate groups and a higher concentration in the region between these locations. Other than this difference, the Na⁺ distributions generated by the two methods largely agreed, as both predicted similar locations of high Na⁺ concentration and nearly identical values of the number of cations and the net solution charge at all distances from the DNA. In contrast, there was greater disagreement between the two methods for Mg²⁺ cation concentration profiles, as both the locations and magnitudes of peaks in Mg²⁺ concentration were different. Despite experimental and simulation observations that Mg²⁺ typically maintains its first solvation shell when interacting with nucleic acids, modeling Mg²⁺ as an unsolvated ion during PB calculations improved the agreement of the Mg²⁺ ion atmosphere predicted by the two methods and allowed for values of the number of bound ions and net solution charge surrounding the DNA from PB calculations that approached the values observed in MD simulations. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 834–848, 2014.     

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg²⁺ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg²⁺ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg²⁺ or Ca²⁺ concentration.     

Mechanisms of gentamicin-induced dysfunction of renal cortical mitochondria: II. Effects on mitochondrial monovalent cation transport

Mitochondrial swelling techniques were used to evaluate the effects of the aminoglycoside antibiotic gentamicin on renal cortical mitochondrial monovalent cation permeability. Gentamicin behaved like EDTA to enhance energy-dependent Na⁺- and K⁺-acetate uptake with a relatively greater effect on Na⁺-acetate uptake. Mg²⁺ prevented and reversed the effects of both EDTA and gentamicin. Neither agent affected energy-independent uptake of Na⁺ and K⁺-acetate. Gentamicin did not enhance energy-independent uptake of K⁺- and Na⁺-nitrate. Gentamicin enhanced energy-dependent swelling in a chloride- and phosphate-containing medium as a function of the medium Na⁺ and K⁺ concentration. This effect occurred simultaneously with gentamicin-induced stimulation of State 4 respiration and was blocked by Mg²⁺. Gentamicin did not affect phosphate transport. The results are taken to indicate a specific action of gentamicin to enhance mitochondrial monovalent cation permeability at an Mg²⁺-sensitive site and it is proposed that this accounts for the effects of gentamicin on mitochondrial respiration.     

Salinomycin: a new monovalent cation ionophore.

The cation discriminations of salinomycin and its derivatives have been studied by measuring complexability with cations and transport rate of them across organic phase. Salinomycin exhibited a great preference for K⁺ over other monovalent and divalent cations in migrating cations into organic phase in two phase systems. The antibiotic mediated the transport of Na⁺ and Rb⁺ as effectively as that of K⁺ across CCl₄ bulk phase, but not those of Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺. From the above results, salinomycin is concluded to act as an alkali ion carrier. The OH-acylated salinomycins retained the activity of parent compound, but the COOH-esterified salinomycins lost the activity.     

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

The uptake and extrusion of monovalent cations by isolated heart mitochondria

Summary The factors involved in the movement of monovalent cations across the inner membrane of the isolated heart mitochondrion are reviewed. The evidence suggests that the energy-dependent uptake of K⁺ and Na⁺ which results in swelling of the matrix is an electrophoretic response to a negative internal potential. There are no clear cut indications that this electrophoretic cation movement is carrier-mediated and possible modes of entry which do not require a carrier are examined. The evidence also suggests that the monovalent cation for proton exchanger (Na⁺ > K⁺) present in the membrane may participate in the energy-dependent extrusion of accumulated ions. The two processes, electrophoretic cation uptake (swelling) and exchange-dependent cation extrusion (contraction) may represent a means of controlling the volume of the mitochondrion within the functioning cell. A number of indications point to the possibility that the volume control process may be mediated by the divalent cations Ca⁺² and Mg⁺². Studies with mercurial reagents also implicate certain membrane thiol groups in the postulated volume control process.An invited article.     

Characterisation of charge distribution and stability of aptamer-thrombin binding interaction

Aptamers are single stranded nucleic acids with specific target-binding functionalities, biophysical and biochemical properties. The binding performance of aptamers to their cognate targets is influenced by the physicochemical conditions of the binding system particularly in relation to biomolecular charge distribution and hydrodynamic conformations in solution. Herein, we report the use of zeta potential measurements to characterise the surface charge distribution, biomolecular hydrodynamic size and the binding performance of a 15-mer thrombin binding aptamer (TBA) to thrombin under various physicochemical conditions of pH, temperature, monovalent (K⁺) and divalent (Mg²⁺) cation concentrations. Charge distribution analysis demonstrated time dependence in the formation of stable TBA-thrombin and TBA-thrombin-metal ion complexes. TBA was characterised to be most stable in pH above 9. The presence of monovalent and divalent metal ions reduced the electronegativity of TBA through electrostatic interactions, and this demonstrated to improve binding characteristics. TBA-thrombin complexes generated under different physicochemical conditions showed varying surface charge distributions. The stability of TBA-thrombin complex investigated using Scatchard analysis showed that the presence of K⁺ increased the binding performance by displaying a positive cooperativity relationship. The presence of Mg²⁺ showed a concave upward trend, potentially caused by heterogeneity in binding.     

Availability of monovalent and divalent cations within intact chloroplasts for the action of ionophores nigericin and A23187

1. A23187 will uncouple electron transport by broken chloroplasts in a divalent cation dependent manner provided that they have been treated with a low concentration of EDTA.2. A23187 stimulates oxaloacetate-dependent oxygen evolution and inhibits phosphoglycerate reduction by intact chloroplasts isolated in a cation-free medium whereas the full effect of nigericin was dependent on the presence of external K⁺.3. Uncoupling of oxaloacetate reduction by A23187 in intact chloroplasts is inhibited by EDTA and this effect is overcome by excess Mg²⁺.4. The results suggest that divalent and not monovalent cations are available for collapsing the light-induced H⁺ gradient within the intact organelle.     

Properties of Plasmamembrane ATPase and Mitochondrial ATPase of Saccharomyces cerevisiae

The properties of mitochondrial ATPase and plasmamembrane ATPase of Saccharomyces cerevisiae are compared. The pH dependence differs considerably. At low pH plasmamembrane ATPase is inactivated. High salt concentrations protect the ATPase against acid inactivation. K⁺ is more effective than Na⁺. The sensitivity of mitochondrial ATPase towards azide, Dio 9 and oligomycin is far greater than found with plasmamembrane ATPase. There are no indications that the membrane ATPase is involved directly either in monovalent cation uptake or in divalent cation uptake. Sr²⁺ and Ca²⁺ do not activate plasmamembrane ATPase and inhibit Mg²⁺-activated ATPase. The substrate specificity of plasmamembrane ATPase is much greater than the substrate specificity of mitochondrial ATPase.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

Two Open States with Progressive Proton Selectivities in the Branched Channelrhodopsin-2 Photocycle

Channelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H⁺. Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H⁺ and divalent cations (Ca²⁺ and/or Mg²⁺), as compared to monovalent cations (Na⁺ and/or K⁺). Compared to monovalent cation conductance, the H⁺ conductance, α, is ∼3 × 10⁶ and the divalent cation conductance, β, is ∼0.01 in the early conducting state. In the stationary mixture of the early and late states, α is larger and β smaller, both by a factor of ∼2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures.