Divalent metal cation binding properties of human prothymosin alpha.

The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.     

Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase

In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.     

Research on the mechanism of interaction between actin and membrane lipids

Using an in vitro system involving pure actin and liposomes, we have established that actin may interact with membrane lipids without any intermediate proteins, and that the mechanism of interaction depends upon the concentration of divalent cation. In the absence of divalent cation, actin increases membrane permeability. Low concentrations (1 mM) of divalent cation potentialize this interaction. In the presence of high divalent cation concentration, actin deposits on the surface of liposomes in a crystalline organization and reduces the membrane microviscosity as shown by the polarization of fluorescence of the DPH probe. We propose that actin interacts with lipids by hydrophobic association which is facilitated by initial electrostatic binding.     

Surface potential of phosphatidylserine monolayers. I. Divalent ion binding effect

Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg²⁺, Ca²⁺ and Mn²⁺) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn²⁺ > Ca²⁺ > Mg²⁺. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane.     

Metal ion binding and the folding of the hairpin ribozyme

The hairpin ribozyme comprises two formally unpaired loops carried on two arms of a four-way helical RNA junction. Addition of divalent metal ions brings about a conformational transition into an antiparallel structure in which there is an intimate association between the loops to generate the active form of the ribozyme. In this study, we have used fluorescence resonance energy transfer to analyze the global folding of the complete ribozyme, and the simple four-way junction derived from it, over a wide concentration range of divalent and monovalent metal ions. The simple junction undergoes an ion-induced rotation into an antiparallel form. In the presence of a constant background concentration of sodium ions, the magnesium-ion-induced transition is characterized by noncooperative binding with a Hill coefficient n = 1. By contrast, the magnesium-ion-induced folding of the complete ribozyme is more complex, involving two distinct binding phases. The first phase occurs in the micromolar range, and involves the cooperative binding of at least three magnesium ions. This can also be achieved by high concentrations of sodium ions, and is therefore likely to be due to diffuse binding of cations at the junction and the interface of the loop-loop interaction. The second phase occurs in the millimolar range, and can only be induced by divalent metal ions. This transition occurs in response to the noncooperative, site-specific binding of magnesium ions. We observe a good correlation between the extent of ion-induced folding and cleavage activity.     

Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.     

Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.     

Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2

Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

Influence of monovalent and divalent cations on the surface area of phosphatidylglycerol monolayers

A theory is presented on the electrostatic properties of the surface area of phosphatidyl-glycerol monolayers spreading at an air-water interface in the presence of monovalent and divalent cations. In the present theory, the adsorption of monovalent and divalent cations to the membranes is taken into account, besides the dissociation of protons, as a possible cause of the change of surface charge density with the variation of pH or ion concentrations. It is also pointed out that, in the presence of structure-making ions such as Li⁺ and Na⁺, the nearest-neighbour interactions between proton dissociation sites become important for the monolayers in the gel state to yield a sharp expansion of the surface area with the increase of pH. The present theory shows quantitative agreements with previously-observed data.     

High-Affinity Cannabinoid Binding Site: Regulation by Ions, Ascorbic Acid, and Nucleotides

The high-affinity cannabinoid site in rat brain is an integral component of brain membranes that recognizes cannabinoids with inhibitory constants (Ki) in the nanomolar range. To clarify its physiological role, we studied the regulation of [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) binding. The site is inhibited by heavy metal ions, such as La3+, at low micromolar concentrations; divalent cations, such as Ca2+ and Mg2+, inhibit [3H]TMA binding, though at somewhat higher concentrations. In contrast, [3H]TMA binding is stimulated by Fe2+, Cu2+, and Hg2+ ions. Ascorbic acid and its analogs are also stimulators of cannabinoid binding at low micromolar concentrations. Stimulation of [3H]TMA binding by ascorbate or ions is dependent upon molecular oxygen, but is not inhibited by metabolic poisons. Metabolically stable nucleoside triphosphate analogs enhance [3H]TMA binding by different mechanisms, with hydrolysis of a high-energy phosphate bond apparently requisite for these influences. These results suggest that the cannabinoid binding site is associated with a nucleotide-utilizing protein possessing multiple regulatory subsites.     

Probing non-selective cation binding in the hairpin ribozyme with Tb(III)

Catalysis by the hairpin ribozyme is stimulated by a wide range of both simple and complex metallic and organic cations. This independence from divalent metal ion binding unequivocally excludes inner-sphere coordination to RNA as an obligatory role for metal ions in catalysis. Hence, the hairpin ribozyme is a unique model to study the role of outer-sphere coordinated cations in folding of a catalytically functional RNA structure. Here, we demonstrate that micromolar concentrations of a deprotonated aqueous complex of the lanthanide metal ion terbium(III), Tb(OH)(aq)(2+), reversibly inhibit the ribozyme by competing for a crucial, yet non-selective cation binding site. Tb(OH)(aq)(2+) also reports a likely location of this binding site through backbone hydrolysis, and permits the analysis of metal binding through sensitized luminescence. We propose that the critical cation-binding site is located at a position within the catalytic core that displays an appropriately-sized pocket and a high negative charge density. We show that cationic occupancy of this site is required for tertiary folding and catalysis, yet the site can be productively occupied by a wide variety of cations. It is striking that micromolar Tb(OH)(aq)(2+) concentrations are compatible with tertiary folding, yet interfere with catalysis. The motif implicated here in cation-binding has also been found to organize the structure of multi-helix loops in evolutionary ancient ribosomal RNAs. Our findings, therefore, illuminate general principles of non-selective outer-sphere cation binding in RNA structure and function that may have prevailed in primitive ribozymes of an early "RNA world".     

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.     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59.The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA.We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.     

Inhibition of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC by Divalent Cations

The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Antimicrobial peptides derived from heme-containing proteins: Hemocidins

Deprived of heme and partially unfolded hemoglobin, myoglobin and cytochrome c display microbicidal activity against a broad spectrum of microorganisms with half maximal lethal dose estimated at micromolar concentrations. The intact proteins were ineffective. Antibacterial activity of these apohemoproteins was also sustained after digestion to approximately 50 amino acids long peptides but further fragmentation abolished microbicidal properties. The most active fragment of apomyoglobin (corresponding to 56–131 region) showed a pronounced effect on the E. coli membrane permeabilization and its action was sensitive to salt as well as to divalent cations concentrations. The membrane-directed effect was specific toward bacteria but no lipopolysaccharide binding properties were observed. No hemolytic properties, even at high peptide concentrations were found; however, a slight but dose-independent cytotoxic effect was observed on fibroblasts and hepatoma cells. The presented data suggest a `carpet-like' mechanism of the membrane-directed activity and may result from exceptional abilities of hemoprotein-derived peptides to form alpha-helical structures. We postulate that the antimicrobial peptides obtained from the heme-containing proteins should be named hemocidins, in contrast to, e.g., hemorphins displaying opioid-like activity.     

Chemical properties of the divalent cation binding site on potassium channels

The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding.