Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 A resolution X-ray crystal structures of Mg(2+) and Ca(2+) salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg(2+) and Ca(2+) to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations' water ligands. In the major groove the cations' water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca(2+) can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg(2+) and Ca(2+) bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg(2+) and Ca(2+) to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg(2+) and Ca(2+) in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg(2+) and Ca(2+) without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.     

Structures of cadmium-binding acidic phospholipase A2 from the venom of Agkistrodon halys Pallas at 1.9A resolution

Phospholipase A(2) coordinates Ca(2+) ion through three carbonyl oxygen atoms of residues 28, 30, and 32, two carboxyl oxygen atoms of residue Asp49, and two (or one) water molecules, forming seven (or six) coordinate geometry of Ca(2+) ligands. Two crystal structures of cadmium-binding acidic phospholipase A(2) from the venom of Agkistrodon halys Pallas (i.e., Agkistrodon blomhoffii brevicaudus) at different pH values (5.9 and 7.4) were determined to 1.9A resolution by the isomorphous difference Fourier method. The well-refined structures revealed that a Cd(2+) ion occupied the position expected for a Ca(2+) ion, and that the substitution of Cd(2+) for Ca(2+) resulted in detectable changes in the metal-binding region: one of the carboxyl oxygen atoms from residue Asp49 was farther from the metal ion while the other one was closer and there were no water molecules coordinating to the metal ion. Thus the Cd(2+)-binding region appears to have four coordinating oxygen ligands. The cadmium binding to the enzyme induced no other significant conformational change in the enzyme molecule elsewhere. The mechanism for divalent cadmium cation to support substrate binding but not catalysis is discussed.     

Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(2).

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca(2+). Effective activation of the C2 domain by intracellular Ca(2+) signals requires high Ca(2+) selectivity to exclude the prevalent physiological metal ions K(+), Na(+), and Mg(2+). The cooperative binding of two Ca(2+) ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg(2+) failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg(2+) did bind but still failed to induce membrane docking. In contrast, Ca(2+), Sr(2+), and Ba(2+) bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyacrylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca(2+), Sr(2+), and Ba(2+) decreased with increasing cation size, suggesting that the C2 domain binds two Ca(2+) or Sr(2+) ions, but only one Ba(2+) ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca(2+) and Sr(2+) triggered docking while even millimolar concentrations of Ba(2+) yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca(2+) ion triggered membrane docking at 20-fold lower concentrations than Sr(2+), due to both the higher Ca(2+) affinity of the free domain and the higher affinity of the Ca(2+)-loaded domain for membranes. Kinetic studies indicated that Ca(2+) ions bound to the free domain are retained at least 5-fold longer than Sr(2+) ions. Moreover, the Ca(2+)-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)-loaded domain. For both Ca(2+) and Sr(2+), the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Ca(2+) selectivity of the C2 domain and suggest that formation of a cPLA(2)-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Characterization of metal ion-binding sites in bacteriorhodopsin

We have investigated the effects of the binding of various metal ions to cation-free bacteriorhodopsin ("blue membrane"). The following have been measured: shift of the absorption maximum from 603 to 558 nm (blue to purple transition), binding isotherms, the release of H+ upon binding, and the decay of the deprotonated intermediate of the photocycle, M412. We find that all cations of the lanthanide series, as well as the alkali and alkali earth metals earlier investigated, are able to bring about the absorption shift, whereas Hg2+ and Pt4+ are not. Sigmoidal spectroscopic titration curves and nonsigmoidal binding curves suggest that there are two high affinity sites for cations in bacteriorhodopsin. Binding to the site with the second highest affinity is responsible for the absorption shift. Divalent cation binding to blue membrane causes release of about six protons, whereas higher numbers of protons are released by trivalent cations, suggesting that the shift of absorption maximum involves proton release from carboxyl group(s). The metal ion bound to this site must be surrounded by carboxyl oxygen atoms acting together as a multidentate ligand with a specific geometry because multivalent ions are effective only when capable of octahedral coordination. Lanthanide ions dramatically inhibit M412 decay at pH above 6.3, an effect probably due to binding to lipid phosphoryl groups.     

Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling

Site-directed spin labeling is used to determine the orientation and depth of insertion of the second C2 domain from synaptotagmin I (C2B) into membrane vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS). EPR line shapes of spin-labeled mutants located with the Ca(2+)-binding loops of C2B broaden in the presence of Ca(2+) and PC/PS vesicles, indicating that these loops undergo a Ca(2+)-dependent insertion into the membrane interface. Power saturation of the EPR spectra provides a position for each spin-labeled site along the bilayer normal, and these EPR-derived distance constraints, along with a high-resolution structure of the C2B domain, are used to generate a model for the domain orientation and position at the membrane interface. Our data show that the isolated C2B domain from synaptotagmin I penetrates PC/PS membranes, and that the backbone of Ca(2+)-binding loops 1 and 3 is inserted below the level of a plane defined by the lipid phosphates. The side chains of several loop residues are within the bilayer interior, and both Ca(2+)-binding sites are positioned near a plane defined by the lipid phosphates. A Tb(3+)-based fluorescence assay is used to compare the membrane affinity of the C2B domain to that of the first synaptotagmin C2 domain (C2A). Both C2A and C2B bind PC/PS (75:25) membrane vesicles with a micromolar lipid affinity in the presence of metal ion. These results indicate that C2A and C2B have a similar membrane affinity and position when bound to PC/PS (75:25) membrane vesicles. EPR spectroscopy indicates that the C2B domain has different interactions with PC/PS membranes containing 1 mol % phosphatidylinositol 4,5-bisphosphate.     

Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.     

Binding Affinity of Metal Ions to the CD11b A-domain Is Regulated by Integrin Activation and Ligands

The divalent cations Mg(2+) and Ca(2+) regulate the interaction of integrins with their cognate ligands, with Mg(2+) uniformly facilitating and Ca(2+) generally inhibiting such interactions in vitro. Because both cations are present in mm concentrations in vivo, the physiologic relevance of the in vitro observations is unclear. We measured the affinity of both cations to the inactive and active states of the ligand- and cation-binding A-domain (CD11bA) from integrin CD11b/CD18 in the absence and presence of the single-chain 107 antibody (scFv107), an activation-insensitive ligand-mimetic antibody. Using titration calorimetry, we found that Mg(2+) and Ca(2+) display equivalent (mm) affinities to inactive CD11bA. Activation induced a approximately 10-fold increase in the binding affinity of Mg(2+) to CD11bA with no change in that of Ca(2+) (106 microm +/- 16 and 2.1 mm +/- 0.19, respectively, n = 4). This increase is largely driven by favorable enthalpy. scFv107 induced a 50-80-fold increase in the binding affinity of Ca(2+) (but not Mg(2+) or Mn(2+)) to either form of CD11bA. Thus the affinity of metal ions to integrins is itself regulated by the activation state of these receptors and by certain ligands. These findings, which we expect will be applicable in vivo, elucidate a new level of regulation of the integrin-metal-ligand ternary complex and help explain some of the discrepant effects of Ca(2+) on integrin-ligand interactions.     

Cation- and peptide-binding properties of human calmodulin-like skin protein

Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with [Ca(2+)](0.5) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) ([Mg(2+)](0.5) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide.     

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.     

Modulation of cardiac ryanodine receptor channels by alkaline earth cations

Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.     

Role of Mg(2+) in Ca(2+)-induced Ca(2+) release through ryanodine receptors of frog skeletal muscle: modulations by adenine nucleotides and caffeine

Mg(2+) serves as a competitive antagonist against Ca(2+) in the high-affinity Ca(2+) activation site (A-site) and as an agonist of Ca(2+) in the low-affinity Ca(2+) inactivation site (I-site) of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). This paper presents the quantitative determination of the affinities for Ca(2+) and Mg(2+) of A- and I-sites of RyR in frog skeletal muscles by measuring [(3)H]ryanodine binding to purified alpha- and beta-RyRs and CICR activity in skinned fibers. There was only a minor difference in affinity at most between alpha- and beta-RyRs. The A-site favored Ca(2+) 20- to 30-fold over Mg(2+), whereas the I-site was nonselective between the two cations. The RyR in situ showed fivefold higher affinities for Ca(2+) and Mg(2+) of both sites than the purified alpha- and beta-RyRs with unchanged cation selectivity. Adenine nucleotides, whose stimulating effect was found to be indistinguishable between free and complexed forms, did not alter the affinities for cations in either site, except for the increased maximum activity of RyR. Caffeine increased not only the affinity of the A-site for Ca(2+) alone, but also the maximum activity of RyR with otherwise minor changes. The results presented here suggest that the rate of CICR in frog skeletal muscles appears to be too low to explain the physiological Ca(2+) release, even though Mg(2+) inhibition disappears.     

Membrane targeting of C2 domains of phospholipase C-delta isoforms.

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca(2+)-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca(2+), Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca(2+)-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca(2+)-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca(2+)-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca(2+)-binding loops and the substitution of Ser for the Ca(2+)-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca(2+) import and non-selective Ca(2+)-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca(2+)-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.     

Activation of 11R-lipoxygenase is fully Ca(2+)-dependent and controlled by the phospholipid composition of the target membrane

Activation of some lipoxygenases (LOX) is found to be related to the selective membrane binding upon cell stimulation. In this study, a systematic analysis of the effect of the lipid composition on the membrane binding efficiency, Ca(2+) affinity, and enzymatic activity of 11R-LOX was performed. The analysis of the membrane targeting by fluorometric and surface plasmon resonance measurements in the absence of Ca(2+) showed an exclusive binding of 11R-LOX to the anionic phospholipids (phosphatidylinositol < phosphatidylglycerol ≈ phosphatidylserine) containing model membranes. The presence of Ca(2+) enhanced the rate of interaction and influenced its mode. The modulation of the activity of 11R-LOX indicated that (i) Ca(2+) binding is a prerequisite for productive membrane association, (ii) the reaction of 11R-LOX with arachidonic acid coincided with and was driven by its Ca(2+)-mediated membrane association, and (iii) phosphatidylethanolamine and anionic phospholipids had a synergistic effect on the Ca(2+) affinity, in line with a target-activated messenger affinity mechanism [Corbin, J. A., et al. (2007) Biochemistry 46, 4322-4336]. According to the mechanism proposed in this report, 11R-LOX can bind to the membranes in two different modes and the efficiency of productive membrane binding is determined by a concerted association of Ca(2+) and lipid headgroups.     

Oxygen binding and its allosteric control in hemoglobin of the primitive branchiopod crustacean Triops cancriformis

Branchiopod crustaceans are endowed with extracellular, high-molecular-mass hemoglobins (Hbs), the functional and allosteric properties of which have largely remained obscure. The Hb of the phylogenetically ancient Triops cancriformis (Notostraca) revealed moderate oxygen affinity, cooperativity and pH dependence (Bohr effect) coefficients: P(50) = 13.3 mmHg, n(50) = 2.3, and Phi = -0.18, at 20 degrees C and pH 7.44 in Tris buffer. The in vivo hemolymph pH was 7.52. Bivalent cations increased oxygen affinity, Mg(2+) exerting a greater effect than Ca(2+). Analysis of cooperative oxygen binding in terms of the nested Monod-Wyman-Changeux (MWC) model revealed an allosteric unit of four oxygen-binding sites and functional coupling of two to three allosteric units. The predicted 2 x 4 and 3 x 4 nested structures are in accord with stoichiometric models of the quarternary structure. The allosteric control mechanism of protons comprises a left shift of the upper asymptote of extended Hill plots which is ascribable to the displacement of the equilibrium between (at least) two high-affinity (relaxed) states, similar to that found in extracellular annelid and pulmonate molluscan Hbs. Remarkably, Mg(2+) ions increased oxygen affinity solely by displacing the equilibrium between the tense and relaxed conformations towards the relaxed states, which accords with the original MWC concept, but appears to be unique among Hbs. This effect is distinctly different from those of ionic effectors (bivalent cations, protons and organic phosphates) on annelid, pulmonate and vertebrate Hbs, which involve changes in the oxygen affinity of the tense and/or relaxed conformations.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca(2+)-ATPase of sarcoplasmic reticulum

The conventional model for transport of Ca(2+) by the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) involves a pair of binding sites for Ca(2+) that change upon phosphorylation of the ATPase from being high affinity and exposed to the cytoplasm to being low affinity and exposed to the lumen. However, a number of recent experiments suggest that in fact transport involves two separate pairs of binding sites for Ca(2+), one pair exposed to the cytoplasmic side and the other pair exposed to the lumenal side. Here we show that the carbodiimide 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC) is membrane-impermeable, and we use EDC to distinguish between cytoplasmic and lumenal sites of reaction. Modification of the Ca(2+)-ATPase in sealed SR vesicles with EDC leads to loss of ATPase activity without modification of the pair of high affinity Ca(2+)-binding sites. Modification of the purified ATPase in unsealed membrane fragments was faster than modification in SR vesicles, suggesting the presence of more quickly reacting lumenal sites. This was confirmed in experiments measuring EDC modification of the ATPase reconstituted randomly into sealed lipid vesicles. Modification of sites on the lumenal face of the ATPase led to loss of the Ca(2+)-induced increase in phosphorylation by P(i). It is concluded that carboxyl groups on the lumenal side of the ATPase are involved in Ca(2+) binding to the lumenal side of the ATPase and that modification of these sites leads to loss of ATPase activity. The presence of MgATP or MgADP leads to faster inhibition of the ATPase by EDC in unsealed membrane fragments than in sealed vesicles, suggesting that binding of MgATP or MgADP to the ATPase leads to a conformational change on the lumenal side of the membrane.