Distances between the functional sites of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase and the lipid/water interface

Measurements of fluorescence energy transfer have been performed to determine the distance between the lipid-water interface and the ATP-binding site in the (Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum. The calculated distance between the donor, FITC bound to the protein (nucleotide binding-site marker), and the acceptor, rhodamine-5′-isothiocyanyldipalmitoylphosphatidylethanolamine (RITC-DPPE) incorporated in the membrane, was in the range of 34–42 Å. In addition the distance between the high affinity Ca²⁺-binding sites and the lipid/water interface has been calculated by luminescence energy transfer from Tb³⁺ bound to the Ca²⁺ sites to RITC-DPPE included in the membrane, and it was approx. 10 Å.     

Molecular structure of troponin C from chicken skeletal muscle at 3Å resolution

Troponin C is the Ca²⁺-binding subunit of the troponin complex and is involved in the calcium control of muscle contraction. The X-ray structure of chicken TnC has been determined at 3Å resolution using a single heavy atom derivative and application of a novel phase improvement and phase extension procedure. The protein has an unusual dumbbell-shape with a length of about 70A. The N- and C-domains are connected by a single long α-helix of about 9 turns. Two metal binding sites (the Ca²⁺-Mg²⁺ sites) in the C-domain are occupied by metal ions in the crystals and the helix-loop-helix Ca²⁺ -binding folds are very similar to those in other known Ca²⁺ -binding proteins. In contrast, the Ca²⁺ -specific sites in the N-domain appear unoccupied and the two putative Ca²⁺ -binding folds have a vastly different structural arrangement. The conformational rearrangements in the N-domain upon Ca²⁺ binding are believed to be the trigger for a cascade of protein-protein interaction alterations which lead to muscle contraction.     

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].     

Structural and energetic parameters of Ca2+ binding to peptides and proteins

The characteristics of Ca²⁺-binding sites and of the structural reorganization induced by Ca²⁺-binding in storage proteins and ion carriers are being studied as models for molecular mechanisms in Ca²⁺ channels and in Ca²⁺-dependent modulatory proteins. A first step in the study was the development of energy parameters for Ca²⁺ compatible with those in the CHARMM package of computer simulation software. Such parameters were obtained from an analytical fit to the potential surface for [(Ca)(OCH₂)₄]²⁺ calculated with an ab initio molecular orbital method. The resulting parametrization was tested for the hexapeptide cyclo-(Pro-Gly)₃, and a 75 residue long calcium binding protein from bovine intestine (ICaBP). The geometrical parameters calculated for the hexapeptide and its 2:1 complex with Ca²⁺ were in good agreement with experimental data from crystallography and nmr. Similarly, the structure of ICaBP optimized with CHARMM using the new Ca²⁺ parameters showed good agreement with the x-ray structure both in the local structures of the calcium-binding sites and in the overall shape of the protein.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: III. A mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca⁺²-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca⁺²-binding site. This site was reconstructed from the fourth potential Ca⁺²-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca⁺²-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca²⁺ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin. For communication II, see [1].     

The effect of calcium (II) on the binding of anticoagulation factor I with activated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca²⁺-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca²⁺ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca²⁺ ions of about 1 mM. The binding of Ca²⁺ ions to ACF I was investigated by equilibrium dialysis and two Ca²⁺-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K₁ and K₂ for these two sites were (1.8 ± 0.5) × 10⁵ and (2.7 ± 0.6) × 10⁴ M^–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca²⁺-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca²⁺ ions. The occupation of both Ca²⁺-binding sites in ACF I required a concentration of Ca²⁺ ions of about 1 mM, which is equal to the effective concentration of Ca²⁺ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca²⁺-binding sites in ACF I with Ca²⁺ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.     

Building reactive copper centers in human carbonic anhydrase II

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu²⁺ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu²⁺-binding events have different affinities (K _a approximately 5 × 10¹² and 1 × 10¹⁰), and both are enthalpically driven processes. Reconstituting these Cu²⁺ sites under a range of conditions has allowed us to assign the Cu²⁺-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu²⁺ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.     

Different Folding Pathways Taken by Highly Homologous Proteins, Goat α-Lactalbumin and Canine Milk Lysozyme

Is the folding pathway conserved in homologous proteins? To address this question, we compared the folding pathways of goat α-lactalbumin and canine milk lysozyme using equilibrium and kinetic circular dichroism spectroscopy. Both Ca²⁺-binding proteins have 41% sequence identity and essentially identical backbone structures. The Φ-value analysis, based on the effect of Ca²⁺ on the folding kinetics, showed that the Ca²⁺-binding site was well organized in the transition state in α-lactalbumin, although it was not yet organized in lysozyme. Equilibrium unfolding and hydrogen-exchange 2D NMR analysis of the molten globule intermediate also showed that different regions were stabilized in the two proteins. In α-lactalbumin, the Ca²⁺-binding site and the C-helix were weakly organized, whereas the A- and B-helices, both distant from the Ca²⁺-binding site, were well organized in lysozyme. The results thus provide an example of highly homologous proteins taking different folding pathways. To understand the molecular origin of this difference, we investigated the native three-dimensional structures of the proteins in terms of non-local contact clusters, a parameter based on the residue-residue contact map and known to be well correlated with the folding rate of non-two-state proteins. There were remarkable differences between the proteins in the distribution of the non-local contact clusters, and these differences provided a reasonable explanation of the observed difference in the folding initiation sites. In conclusion, the protein folding pathway is determined not only by the backbone topology but also by the specific side-chain interactions of contacting residues.     

NMR structure and calcium-binding properties of the tellurite resistance protein TerD from Klebsiella pneumoniae

The tellurium oxyanion TeO₃²⁻ has been used in the treatment of infectious diseases caused by mycobacteria. However, many pathogenic bacteria show tellurite resistance. Several tellurite resistance genes have been identified, and these genes mediate responses to diverse extracellular stimuli, but the mechanisms underlying their functions are unknown. To shed light on the function of KP-TerD, a 20.5 -kDa tellurite resistance protein from a plasmid of Klebsiella pneumoniae, we have determined its three-dimensional structure in solution using NMR spectroscopy. KP-TerD contains a β-sandwich formed by two five-stranded β-sheets and six short helices. The structure exhibits two negative clusters in loop regions on the top of the sandwich, suggesting that KP-TerD may bind metal ions. Indeed, thermal denaturation experiments monitored by circular dichroism and NMR studies reveal that KP-TerD binds Ca²⁺. Inductively coupled plasma-optical emission spectroscopy shows that the binding ratio of KP-TerD to Ca²⁺ is 1:2. EDTA (ethylenediaminetetraacetic acid) titrations of Ca²⁺-saturated KP-TerD monitored by one-dimensional NMR yield estimated dissociation constants of 18  and 200 nM for the two Ca²⁺-binding sites of KP-TerD. NMR structures incorporating two Ca²⁺ ions define a novel bipartite Ca²⁺-binding motif that is predicted to be highly conserved in TerD proteins. Moreover, these Ca²⁺-binding sites are also predicted to be present in two additional tellurite resistance proteins, TerE and TerZ. These results suggest that some form of Ca²⁺ signaling plays a crucial role in tellurite resistance and in other responses of bacteria to multiple external stimuli that depend on the Ter genes.     

Terbium(III) fluorescence probe studies on metal ion-binding sites in anticoagulation factor I from Agkistrodon acutus venom

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that holo-ACF I assumes a compactly folded structure in the range of pH 5–6, in which the most interior Trp residues and quenchers are adjacent. Tb³⁺ ions can completely replace both Ca²⁺ ions in holo-ACF I, as determined by equilibrium dialysis. Although the two Tb³⁺ ions in Tb³⁺-ACF I have slightly different luminescence efficiencies, both have similar quenching effects on the intrinsic fluorescence, suggesting that probably there are same numbers of Trp residues close to both Tb³⁺-binding sites. Two Tb³⁺-binding sites with similar apparent Tb³⁺ association constant values, (1.69 ± 0.02) × 10⁷ M^–1 and (1.42 ± 0.01) × 10⁷ M^–1, respectively, were further identified through Tb³⁺ fluorescence titration. In addition, it has been confirmed from the titration of holo-ACF I and Tb³⁺-ACF I with NBS that only interior Trp residues are involved in the energy transfer to Tb³⁺ ions and that all accessible Trp residues located in the surface of holo-ACF I have similar affinity to NBS, while those located in the surface of Tb³⁺-ACF I have two different kinds of affinity to NBS, which strongly suggests a conformational change of holo-ACF I upon substitution of Tb³⁺ for Ca²⁺. The results show that although the Tb³⁺-altered conformation of ACF I cannot support the binding of Tb³⁺-ACF I with FXa, determined by nondenaturing PAGE, Tb³⁺ ions are effective and useful fluorescence probes to analyze the structures and properties of Ca²⁺-binding sites in ACF I.     

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Statistical analysis of structural characteristics of protein Ca<Superscript>2+</Superscript>-binding sites

To better understand the biological significance of Ca²⁺, we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca²⁺-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 ± 2 vs. 7 ± 1), fewer protein ligands (4 ± 2 vs. 6 ± 1), and more water ligands (2 ± 2 vs. 1 ± 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H₂O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H₂O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 ± 1 vs. 3 ± 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix–loop–helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca–O–C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca²⁺-binding proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Michael Kirberger, Xue Wang, and Hai Deng contributed equally to this article.     

Towards predicting Ca2+‐binding sites with different coordination numbers in proteins with atomic resolution

Ca²⁺‐binding sites in proteins exhibit a wide range of polygonal geometries that directly relate to an equally‐diverse set of biological functions. Although the highly‐conserved EF‐Hand motif has been studied extensively, non‐EF‐Hand sites exhibit much more structural diversity which has inhibited efforts to determine the precise location of Ca²⁺‐binding sites, especially for sites with few coordinating ligands. Previously, we established an algorithm capable of predicting Ca²⁺‐binding sites using graph theory to identify oxygen clusters comprised of four atoms lying on a sphere of specified radius, the center of which was the predicted calcium position. Here we describe a new algorithm, MUG (MUltiple Geometries), which predicts Ca²⁺‐binding sites in proteins with atomic resolution. After first identifying all the possible oxygen clusters by finding maximal cliques, a calcium center (CC) for each cluster, corresponding to the potential Ca²⁺ position, is located to maximally regularize the structure of the (cluster, CC) pair. The structure is then inspected by geometric filters. An unqualified (cluster, CC) pair is further handled by recursively removing oxygen atoms and relocating the CC until its structure is either qualified or contains fewer than four ligand atoms. Ligand coordination is then determined for qualified structures. This algorithm, which predicts both Ca²⁺ positions and ligand groups, has been shown to successfully predict over 90% of the documented Ca²⁺‐binding sites in three datasets of highly‐diversified protein structures with 0.22 to 0.49 Å accuracy. All multiple‐binding sites (i.e. sites with a single ligand atom associated with multiple calcium ions) were predicted, as were half of the low‐coordination sites (i.e. sites with less than four protein ligand atoms) and 14/16 cofactor‐coordinating sites. Additionally, this algorithm has the flexibility to incorporate surface water molecules and protein cofactors to further improve the prediction for low‐coordination and cofactor‐coordinating Ca²⁺‐binding sites. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca²⁺, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca²⁺, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [³H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca²⁺-binding site to sensitize RyR1 to Ca²⁺. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca²⁺. However, enhanced RyR1 activation occurred in the presence of Ca²⁺, AMPPCP, and Caf. In the absence of Ca²⁺, Na⁺ inhibited [³H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca²⁺-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca²⁺, Na⁺ is predicted to inhibit RyR1 by interacting with the Ca²⁺-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca²⁺-binding site, and conversely, Ca²⁺ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca²⁺, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca²⁺-, ATP-, and Caf-binding sites.     

Voltage control of Ca2+ permeation through N-type calcium (CaV2.2) channels

Voltage-gated calcium (Ca_V) channels deliver Ca²⁺ to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca²⁺ over other cations is thought to involve multiple Ca²⁺-binding sites within the pore. Although the Ca²⁺ affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca_V2.2) channels to investigate the effect of voltage on Ca²⁺ flux. We found that the EC₅₀ for Ca²⁺ permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca²⁺ ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca_V2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca²⁺-Ba²⁺ anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca²⁺-Ba²⁺ anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca²⁺ permeation through Ca_V2.2 channels may require at least four Ca²⁺-binding sites. Finally, our results suggest that the high affinity of Ca²⁺ for the channel helps to enhance Ca²⁺ influx at depolarized voltages relative to other ions (e.g., Ba²⁺ or Na⁺), whereas the absence of voltage effects at negative potentials prevents Ca²⁺ from becoming a channel blocker. Both effects are needed to maximize Ca²⁺ influx over the voltages spanned by action potentials.     

Calcium transport by rabbit skeletal muscle microsomes (“fragmented sarcoplasmic reticulum”)

Ca²⁺ binding by skeletal muscle microsomes in 5 mM ATP exhibited saturation kinetics in the range of Ca²⁺ concentrations between 3 · 10^?8 and 10^?5 M. Approximately 140 nmoles binding sites per mg protein were found. These had a Ca²⁺ binding constant of approximately 4.5 · 10⁶ M^?1 with half saturation at 2.2 · 10^?7 M Ca²⁺. In the presence of oxalate, much larger amounts of Ca²⁺ (approx. 6 μmoles/mg protein) were taken up by the microsomes (Ca²⁺ uptake), but the rate of Ca²⁺ uptake increased linearly with [Ca²⁺] when ionized Ca²⁺ concentrations were below 3 · 10^?6 M. At Ca²⁺ concentrations above 3 · 10^?6, Ca²⁺ uptake was inhibited. Double reciprocal plots of the Ca²⁺ dependence of the initial rates of Ca²⁺ uptake in the concentration range between 3 · 10^?7 M and 10^?5 M, unlike those of Ca²⁺ binding, did not demonstrate saturation kinetics, but could be interpreted as representing a non-saturable system with inhibition at higher Ca²⁺ concentrations. In view of these differences, and because Ca²⁺-binding sites were almost fully saturated at 10^?6 M Ca²⁺, whereas Ca²⁺ uptake rate increased linearly with increasing [Ca²⁺] to approximately 3 · 10^?6 M, the Ca²⁺-binding sites are not shown kinetically to participate in oxalate-dependent Ca²⁺ uptake.     

The determination of the separation of tyrosine-99 and tyrosine-138 in calmodulin: Radiationless energy transfer

The average separation of the phenolic groups of tyrosine-99 and tyrosine-138 has been measured by radiationless energy transfer between each tyrosine and the nitro derivative of the second tyrosine. A separation of 16.7 ± 0.7 Å was found in the absence of Ca²⁺ and 15.5 ± 0.7 Å in the presence of Ca²⁺.     

Infrared spectroscopic study of the metal-coordination structures of calcium-binding proteins

Carboxylate (COO⁻) groups can coordinate to metal ions in of the following four modes: ‘unidentate’, ‘bidentate’, ‘bridging’ and ‘pseudo-bridging’ modes. COO⁻ stretching frequencies provide information about the coordination modes of COO⁻ groups to metal ions. We review the Fourier-transform infrared spectroscopy (FTIR) of side-chain COO⁻ groups of Ca²⁺-binding proteins: pike parvalbumin pI 4.10, bovine calmodulin and Akazara scallop troponin C. FTIR spectroscopy of Akazara scallop troponin C has demonstrated that the coordination structure of Mg²⁺ is distinctly different from that of Ca²⁺ in the Ca²⁺-binding site. The assignments of the COO⁻ antisymmetric stretch have been ensured on the basis of the spectra of calcium-binding peptide analogues. The downshift of the COO⁻ antisymmetric stretching mode from 1565 cm^-1 to 1555-1540 cm⁻¹ upon Ca²⁺ binding is a commonly observed feature of FTIR spectra for EF-hand proteins.     

The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca²⁺ binding in the absence of added Mg²⁺ to the purified sarcoplasmic reticulum Ca²⁺-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca²⁺-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca²⁺ binding was 4·10⁵ M^?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH₂]PP has little effect on the Ca²⁺-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca²⁺ binding. ADP decreased the level of ATP-induced Ca²⁺ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca²⁺ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca²⁺ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl₂; after the release of Ca²⁺, the phosphoenzyme decayed. These observations suggest that Mg²⁺, added after the ATP-induced Ca²⁺-binding process, may replace the Ca²⁺ on the phosphoenzyme and initiate phosphoenzyme decomposition.     

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.