Point amino acid substitutions in the Ca2+-binding sites of recoverin: II. The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca²⁺-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca²⁺ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability (its thermal transition shifts by 15°C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca²⁺ are also characteristic of the -EF2 mutant of recoverin whose second Ca²⁺-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca²⁺-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions. For the communication I, see [1].     

Effect of Mg<Superscript>2+</Superscript> versus Ca<Superscript>2+</Superscript> on the behavior of Annexin A5 in a membrane-bound state

Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca²⁺ dependent manner. Several studies already demonstrate that Mg²⁺ ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca²⁺ versus Mg²⁺ on AnxA5 binding to membrane models. In the presence of Ca²⁺, AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca²⁺ ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg²⁺, instead of Ca²⁺, no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca²⁺ ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg²⁺ ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg²⁺ ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.     

R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg²⁺. Surprisingly, Ca²⁺ suppresses the Mg²⁺-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca²⁺-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg²⁺- and Mn²⁺-mediated reactions and was about three times slower with Ca²⁺. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg²⁺-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

The role of Ca2+ in the activity of Mycobacterium tuberculosis DNA gyrase

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca²⁺-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca²⁺ cannot support topoisomerase reactions in the absence of Mg²⁺, but partial removal of Ca²⁺ from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca²⁺. More extensive removal of Ca²⁺ by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca²⁺-binding residues also leads to loss of activity. We propose that Ca²⁺ has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca²⁺ binding.     

Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca²⁺-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca²⁺-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr²⁺ and Ba²⁺, with ionic radii larger than the ionic radius of Ca²⁺, can bind to Ca²⁺-free ACF I (apo-ACF I), while Mg²⁺, with an ionic radius smaller than that of Ca²⁺, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr²⁺-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca²⁺–ACF I, while Ba²⁺-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca²⁺–ACF I. Mg²⁺ and Sr²⁺, with ionic radii close to that of Ca²⁺, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba²⁺, with a much larger ionic radius than Ca²⁺, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg²⁺, Ca²⁺, and Sr²⁺ ions to FXa may be essential for the recognition between FXa and ACF I.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

Restriction endonucleases of the PD…D/EXK family need Mg²⁺ for DNA cleavage. Whereas Mg²⁺ (or Mn²⁺) promotes catalysis, Ca²⁺ (without Mg²⁺) only supports DNA binding. The role of Mg²⁺ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg²⁺ involved in catalysis. To address this problem, we measured the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me²⁺ per active site. DNA cleavage experiments were carried out at various Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg²⁺ and Mn²⁺ concentration dependence. In general, the Mg²⁺ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn²⁺ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg²⁺ or Mn²⁺ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca²⁺. Based on these results, we propose that one Mg²⁺ or Mn²⁺ is critical for restriction enzyme activation, and binding of a second Me²⁺ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg²⁺ or Mn²⁺ mainly leads to an increase in K_m, such that the inhibitory effect of excess Mg²⁺ or Mn²⁺ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me²⁺ binding to these enzymes.     

Control of insulin receptor affinity by a Ca2+-sensitive binding site

Calcium (Ca²⁺) increased insulin-receptor binding in both membrane and solubilised receptor preparations. Ca²⁺ increased both receptor affinity and initial rate of association of [¹²⁵I]insulin to the receptor preparations. Ca²⁺ had no effect on insulin receptor number in either receptor preparation. The effect of Ca²⁺ on affinity could be mimicked by ions with similar ionic radii and properties (e.g., Ba²⁺, Mg²⁺ and Sr²⁺). EDTA and oleic acid reduced insulin binding and receptor affinity and these effects were reversed by the addition of Ca²⁺. These studies suggest that Ca²⁺ and Ca²⁺-like ions may bind to a site on or near the receptor and may be responsible for a conformational change with a consequent increase in receptor affinity.     

Functional Contribution of Ca2+ and Mg2+ to the Intermolecular Interaction of Visinin-like Proteins

The interaction of human visinin-like protein 1 (VILIP1) and visinin-like protein 3 (VILIP3) with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺) was explored using circular dichroism and fluorescence measurement. These results showed that the four cations each induced a different subtle change in the conformation of VILIPs. Moreover, VILIP1 and VILIP3 bound with Ca²⁺ or Mg²⁺ in a cooperative manner. Studies on the truncated mutants showed that the intact EF-3 and EF-4 were essential for the binding of VILIP1 with Ca²⁺ and Mg²⁺. Pull-down assay revealed that Ca²⁺ and Mg²⁺ enhanced the intermolecular interaction of VILIPs, and led to the formation of homo- and hetero-oligomer of VILIPs. Together with previous findings that Ca²⁺-dependent localization of VILIPs may be involved in the regulation of distinct cascades and deprivation of Ca²⁺-binding capacity of VILIPs did not completely eliminate their activity, it is likely to reflect that Mg²⁺-bound VILIPs may play a role in regulating the biological function of VILIPs in response to a concentration fluctuation of Ca²⁺ in cells.     

DNA distortion and specificity in a sequence-specific endonuclease

Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca²⁺, Mg²⁺, and Mn²⁺) are presented. While previous structures were produced from soaking Ca²⁺ into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca²⁺, Mg²⁺, or Mn²⁺. The Mn²⁺-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca²⁺ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg²⁺) and calcium (Ca²⁺) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3′ to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg²⁺ ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K⁺) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K_d) for TATA increases in the order K⁺ < Na⁺ < Ca²⁺ < Mg²⁺. Except for Mg²⁺, K_d for TATA relative to ATAT is independent of ion concentration, whereas for Mg²⁺ the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg²⁺, additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.     

Analysis of Ca2+/Mg2+ selectivity in α‐lactalbumin and Ca2+‐binding lysozyme reveals a distinct Mg2+‐specific site in lysozyme

The triggering of Ca²⁺ signaling pathways relies on Ca²⁺/Mg²⁺ specificity of proteins mediating these pathways. Two homologous milk Ca²⁺‐binding proteins, bovine α‐lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest “four‐state” scheme of metal‐ and temperature‐induced structural changes in a protein. The association of Ca²⁺/Mg²⁺ by native proteins is entropy‐driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca²⁺ and Mg²⁺, due to low thermal stabilities of their apo‐forms and relatively high unfavorable enthalpies of Mg²⁺ association. The ratios of their apparent affinities to Ca²⁺ and Mg²⁺, being unusually high at low temperatures (5.3–6.5 orders of magnitude), reach the values inherent to classical EF‐hand motifs at physiological temperatures. The comparison of phase diagrams predicted within the model of competitive Ca²⁺ and Mg²⁺ binding with experimental data strongly suggests that the association of Ca²⁺ and Mg²⁺ ions with bLA is a competitive process, whereas the primary Mg²⁺ site of EQL is different from its Ca²⁺‐binding site. The later conclusion is corroborated by qualitatively different molar ellipticity changes in near‐UV region accompanying Mg²⁺ and Ca²⁺ association. The Ca²⁺/Mg²⁺ selectivity of Mg²⁺‐site of EQL is below an order of magnitude. EQL exhibits a distinct Mg²⁺‐specific site, probably arising as an adaptation to the extracellular environment. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interaction of Na+ and Mg2+ with Ca2+ in pancreatic islets as visualized by chlorotetracycline fluorescence

The fluorescence of microdissected pancreatic islets of ob/ob-mice was studied by microscope photometry after incubation with 10 μM chlorotetracycline. In Krebs-Ringer bicarbonate buffer, excitation at 390 nm yielded peak emission at 530 nm, suggesting that chelated Ca²⁺ was the major source of fluorescence. In support of this interpretation, incubation in Ca²⁺-free buffer markedly decreased the fluorescence, whereas withdrawal of Mg²⁺ increased it. Raising the Mg²⁺ concentration to 15 mM suppressed the fluorescence. In the presence of Ca²⁺, the substitution of choline ions for Na⁺ increased the fluorescence considerably; in the absence of Ca²⁺, however, Na⁺ deficiency had only little effect. Control experiments showed that Na⁺ or choline ions had no effect on the fluorescence of Ca²⁺-chlorotetracycline in 70 or 90% methanol. In 90%, but not in 70%, methanol 15 mM Mg²⁺ slightly quenched the fluorescence from 2.5 mM Ca²⁺ and 10 μM chlorotetracycline. It is suggested that Na⁺, and perhaps Mg²⁺, tends to decrease the amount of membrane-bound Ca²⁺ in the pancreatic islets.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

Microsecond Molecular Dynamics Simulations of Mg2+- and K+- Bound E1 Intermediate States of the Calcium Pump

We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca²⁺-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg²⁺ in the absence of Ca²⁺ has shown that SERCA adopts an E1 structure with transmembrane Ca²⁺-binding sites I and II exposed to the cytosol, stabilized by a single Mg²⁺ bound to a hybrid binding site I′. This Mg²⁺-bound E1 intermediate state, designated E1•Mg²⁺, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca²⁺ by facilitating H⁺/Ca²⁺ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg²⁺ crystal structure, starting in the presence or absence of initially-bound Mg²⁺. Both simulations were performed for 1 µs in a solution containing 100 mM K⁺ and 5 mM Mg²⁺ in the absence of Ca²⁺, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg²⁺, SERCA site I′ maintained Mg²⁺ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg²⁺, two K⁺ ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg²⁺ and E•2K⁺ intermediate states of SERCA are populated in solution in the absence of Ca²⁺, with the more open 2K⁺-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K⁺ state acts as a functional intermediate that facilitates the E2 to E1•2Ca²⁺ transition through two mechanisms: by pre-organizing transport sites for Ca²⁺ binding, and by partially opening the cytosolic headpiece prior to Ca²⁺ activation of nucleotide binding.     

Conformation and reactivity of DNA. IV. Base binding ability of transition metal ions to native DNA and effect on helix conformation with special reference to DNA-Zn(II) complex

The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements. At low ionic strength (10^?3 M NaClO₄) DNA shows a maximum in the difference absorption spectra in the presence of Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺, and Ni²⁺ but not with Mg²⁺ or Ca²⁺. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn²⁺ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn²⁺ per DNA phosphate at 10^?3 M NaClO₄; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA. Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg²⁺ or Ca²⁺. The data are interpreted in terms of a preferential interaction of Zn²⁺ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn²⁺ to DNA disappears between 0.5 M and 1 M NaClO₄, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO₄ (3?7.2 M NaClO₄) or at 0.5 to 2 M Mn²⁺. At relatively high cation concentration Mg²⁺ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn²⁺, Zn²⁺ on one hand and Mg²⁺ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg²⁺, Ba²⁺, < Ca²⁺ < Fe²⁺, Ni²⁺, Co²⁺ < Mn²⁺, Zn²⁺ < Cd²⁺ < Cu²⁺. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn²⁺ and Mn²⁺ in DNA synthesis reported in the literature.     

Sorption of alkaline earth metal ions Ca2+ and Mg2+ on lyocell fibres

Ca²⁺ and Mg²⁺ content of cellulose fibres is of relevance for a wide range of applications e.g. textile processing, pulp/paper, food. Sorption of Ca²⁺ and Mg²⁺ ions were found on lyocell type regenerated cellulose fibres. Higher affinity was found for Ca²⁺ ions compared to Mg²⁺ ions. At pH 9, fibre saturation was observed at a calcium binding capacity of 18–20 mmol/kg. A carboxylic group content of 18 mmol COOH per kg fibre material was determined based on the Methylene Blue absorption. This indicates a 1:1 molar stoichiometry between the carboxylic groups present in the fibres and the bound Ca²⁺ ions. Thus it is proposed that the salt in fibre shows the general composition (Cell-O^? Ca²⁺ X^?), X^? being an anion bound in the salt to achieve charge neutrality.The sorption of Ca²⁺ also can be demonstrated by complex formation with 1,2-dihydroxy-9,10-anthraquinone (alizarin) which forms a red-violet Ca²⁺-complex. Colour fixation thus can be used as an indicator for the Ca²⁺-ions bound in the fibre.     

Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+

It is known that extracellular Mg²⁺ and Ca²⁺ can permeate TRPM7 and at the same time block the permeation by monovalent cations. In the present study, we examined the molecular basis for the conductivity and sensitivity of human TRPM7 to these divalent cations. Extracellular acidification to pH 4.0 markedly reduced the blocking effects of Mg²⁺ and Ca²⁺ on the Cs+ currents, decreasing their binding affinities: their IC50 values increased 510- and 447-fold, respectively. We examined the effects of neutralizing each of four negatively charged amino acid residues, Glu-1047, Glu-1052, Asp-1054 and Asp-1059, within the putative pore-forming region of human TRPM7. Mutating Glu-1047 to alanine (E1047A) resulted in non-functional channels, whereas mutating any of the other residues resulted in functionally expressed channels. Cs+ currents through D1054A and E1052A were less sensitive to block by divalent cations; the IC50 values were increased 5.5- and 3.9-fold, respectively, for Mg2+ and 10.5- and 6.7-fold, respectively, for Ca2+. D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg2+ (1.7-fold) and Ca2+ (3.9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg²⁺ and Ca²⁺. In the E1052A and D1059A mutants, inward Mg²⁺ and Ca²⁺ currents were sizable but significantly diminished. Thus, it is concluded that in human TRPM7, (1) both Asp-1054 and Glu-1052, which are located near the narrowest portion in the pore's selectivity filter, may provide the binding sites for Mg²⁺ and Ca²⁺, (2) Asp-1054 is an essential determinant of Mg²⁺ and Ca²⁺ conductivity, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.