SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

The sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca²⁺. This directed energy transfer requires cross‐talk between the two Ca²⁺ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu³⁰⁹ contributes to Ca²⁺ coordination at site II, and a consensus has been that E309Q only binds Ca²⁺ at site I. The crystal structure of E309Q in the presence of Ca²⁺ and an ATP analogue, however, reveals two occupied Ca²⁺ sites of a non‐catalytic Ca₂E1 state. Ca²⁺ is bound with micromolar affinity by both Ca²⁺ sites in E309Q, but without cooperativity. The Ca²⁺‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca²⁺ site II.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Docking of calcium ions in proteins with flexible side chains and deformable backbones

A method of docking Ca²⁺ ions in proteins with flexible side chains and deformable backbones is proposed. The energy was calculated with the AMBER force field, implicit solvent, and solvent exposure-dependent and distance-dependent dielectric function. Starting structures were generated with Ca²⁺ coordinates and side-chain torsions sampled in 1000 Å³ cubes centered at the experimental Ca²⁺ positions. The energy was Monte Carlo-minimized. The method was tested on fourteen Ca²⁺-binding sites. For twelve Ca²⁺-binding sites the root mean square (RMS) deviation of the apparent global minimum from the experimental structure was below 1.3 and 1.7 Å for Ca²⁺ ions and side-chain heavy atoms, respectively. Energies of multiple local minima correlate with the RMS deviations from the X-ray structures. Two Ca²⁺-binding sites at the surface of proteinase K were not predicted, because of underestimation of Ca²⁺ hydration energy by the implicit-solvent method.     

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⁺.     

Inhibition of erythrocyte membrane (Ca2+ + Mg2+)-ATPase by the organophosphorus insecticides parathion and methylparathion

Organophosphorus insecticides parathion and methylparathion non-competitively inhibited the activity of (Ca²⁺ + Mg²⁺)-ATPase bound to and solubilized from pig erythrocyte membrane. Both enzyme preparations exhibited biphasic substrate curves displaying the existence of two functional active sites with low and high affinity to ATP. Also, the relationship between the activity of bound enzyme and Ca²⁺ concentration was biphasic. The activity reached maximum at 20 μM then dropped progressively as the Ca²⁺ concentration was raised. The inhibition of the activity was more pronounced for parathion than for methylparathion and the solubilized enzyme preparation was more affected than the bound one. The inhibition constants (K_i) for parathion for bound enzyme were 55 and 158 μM for high- and low-affinity active sites, respectively; for methylparathion these values equalled 74 and 263 μM, respectively. K_i values for parathion were 36 and 118 μM for solubilized enzyme (high- and low-affinity sites, respectively), for methylparathion −62 and 166 μM, respectively. The magnitude of the effect was greater for a low Ca²⁺ concentration, which could arise from different conformational states of the enzyme at different calcium concentrations. The results of the experiment suggest that the insecticides inhibited the ATPase by binding to a site on the enzyme rather than by the interaction with associated lipids, although lipids could weaken the action of the compounds due to the strong affinity of organophosphorus insecticides to lipids.     

Tertiary structure and energy coupling in Ca2+-pump system

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca²⁺ Mg²⁺)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H₂O molecules surrounding the Ca²⁺ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca²⁺ - Ca²⁺ intersite distance is estimated to be 8–9 Å and the average distance from the Ca²⁺ sites to CrATP is about 18 Å.Digestion of the (Ca²⁺ + Mg²⁺)-ATPase at the T₂ site (Arg 198) causes uncoupling of Ca²⁺-transport from ATPase activity while calcium occlusion due to E₁-P formation remains unchanged. Further tryptic digestion beyond T₂ and in the presence of ATP diminishes Ca²⁺ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T₂ and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca²⁺ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.     

Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA)

The transfer of Ca²⁺ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca²⁺ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca²⁺ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca²⁺ uptake pathway or establish distinct routes for mitochondrial Ca²⁺ sequestration. In this study, we show that a modulation of Ca²⁺ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca²⁺ signals and consequently switches mitochondrial Ca²⁺ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca²⁺ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca²⁺ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca²⁺ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca²⁺ rises.     

Phosphoprotein intermediate in the ca2+-dependent atpase reaction of macrophage plasma membrane

ATPase activity and phosphorylation by [γ-³²P] ATP of isolated plasma membrane of alveolar macorphages are stimulated in a parallel fashion by physiologic concentrations of Ca²⁺, with half-maximal activating effect of this ion at (3–7) × 10^?7 M. For various membrane preparations, a direct proportionality exists between Ca²⁺-dependent ATPase activity and amount of ³²P incorporated. Labeling of membrane attains the steady-state level by 10 sec at 0°C, and is rapidly reversed by adenosine diphosphate (ADP). K⁺ decreases the amount of membrane-bound ³²P, mainly by enhancing the rate of dephosphorylation of the ³²P-intermediate. Hydroxylamine causes a release of about 90% of ³²P bound to the membrane, thus indicating that the ³²P-intermediate contains an acyl-phosphate bond. When the labeled plasma membrane is solubilized and electrophoresed on acrylamide gels in the presence of sodium dodecyl sulphate, the radioactivity appears to be largely associated with a single protein fraction of 132,500 ± 2,000 apparent molecular weight. These features of the macrophage Ca²⁺-ATPase suggest that the enzyme activity might be part of a surface-localized Ca²⁺-extrusion system, participating in the regulation of Ca²⁺-dependent activities of the macrophage.     

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn²⁺ and Ca²⁺. These experiments were performed to find the Mn²⁺ nearest-neighbor distance and thereby determine whether carboxylate-Mn²⁺ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn²⁺ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca²⁺ was found to reduce the Mn²⁺ spin-spin line broadening at similar total bound Mn²⁺ concentrations. This is expected only if Ca²⁺ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.     

Role of the Sarcoplasmic Reticulum Ca2+-ATPase on Heat Production and Thermogenesis

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca²⁺-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca²⁺ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca²⁺ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca²⁺ transport and the fraction which is dissipated in the surrounding medium as heat.     

Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca(2+) -bound closed state: Implications for target recognition

Calcium‐binding protein 1 (CaBP1), a neuron‐specific member of the calmodulin (CaM) superfamily, regulates the Ca²⁺‐dependent activity of inositol 1,4,5‐triphosphate receptors (InsP3Rs) and various voltage‐gated Ca²⁺ channels. Here, we present the NMR structure of full‐length CaBP1 with Ca²⁺ bound at the first, third, and fourth EF‐hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root‐mean‐squared deviation of 0.54 Å (N‐domain) and 0.48 Å (C‐domain). The first 18 residues from the N‐terminus in CaBP1 (located upstream of the first EF‐hand) are structurally disordered and solvent exposed. The Ca²⁺‐saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N‐domain structure of CaBP1 contains two EF‐hands (EF1 and EF2), both in a closed conformation [interhelical angles = 129° (EF1) and 142° (EF2)]. The C‐domain contains EF3 and EF4 in the familiar Ca²⁺‐bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N‐domain adopts the same closed conformation in the presence or absence of Ca²⁺ bound at EF1. The Ca²⁺‐bound closed conformation of EF1 is reminiscent of Ca²⁺‐bound EF‐hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca²⁺‐bound closed conformation of EF1 in CaBP1 might undergo an induced‐fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.     

Effect of Mg2+ and spermine on the kinetics of Ca2+ transport in rat-liver mitochondria

Plots relating the initial rate of mitochondrial Ca²⁺ transport to the Ca²⁺ concentration (kinetic plots) have a hyperbolic shape in a Ca²⁺ concentration range of 2.5–100 µM as measured in sucrose or KCl media. In the presence of Mg²⁺ or a polyamine spermine, which both are competitive inhibitors of Ca²⁺ binding to low affinity sites at the membrane surface, the shape of the plots becomes sigmoidal. At higher concentrations of these agents linear kinetic plots are obtained as measured in a sucrose medium. In a KCl medium the sigmoidality of the kinetic plots is enhanced by an increase in the Mg²⁺ or spermine concentration. It is suggested that Mg²⁺ and spermine affect the kinetics of Ca²⁺ transport by interfering with Ca²⁺ binding to low affinity sites of the membrane surface and that the binding of Ca²⁺ to these sites is the first step of the mitochondrial Ca²⁺ transport.     

The interaction of La 3+ with mitochondria in relation to respiration-coupled Ca 2+ transport

La³⁺ inhibits the respiration-dependent accumulation of Ca²⁺ by rat liver mitochondria when added in very small amounts (0.1–l.0 nmole per mg protein). However, La³⁺ itself does not activate respiration. With the use of ¹⁴⁰La³⁺ it was found that La³⁺ is very rapidly bound to rat liver mitochondria in a respiration-independent process accompanied by loss of H⁺ to the medium. When both La³⁺ and Ca²⁺ are added to mitochondria simultaneously, most of the La³⁺ but little Ca²⁺ are bound. La³⁺ added to mitochondria previously loaded with Ca²⁺ is tightly bound without discharge of Ca²⁺. Conversely, when Ca²⁺ is added to La³⁺-loaded mitochondria it is not bound nor is the La³⁺ discharged. La³⁺ inhibits both high-affinity and low-affinity respiration-independent Ca²⁺ binding. Isotopic experiments showed that La³⁺ is, in fact, bound to the same high-affinity sites as Ca²⁺, in both intact mitochondria and in mitochondrial extracts. It is concluded (1) that La³⁺ binds to and inhibits the Ca²⁺ carrier; (2) that La³⁺ is not transported by the Ca²⁺ carrier; and (3) that La³⁺ is, in addition, bound to a large number of external sites on mitochondria for which Ca²⁺ is not a strong competitor.     

Mitochondrial Colocalization with Ca2+ Release Sites is Crucial to Cardiac Metabolism

In cardiomyocyte subcellular structures, colocalization of mitochondria with Ca²⁺ release sites is implicated in regulation of cardiac energetics by facilitating Ca²⁺ influx into mitochondria to modulate the tricarboxylic acid (TCA) cycle. However, current experimental techniques limit detailed examination of this regulatory mechanism. Earlier, we developed a three-dimensional (3D) finite-element cardiomyocyte model featuring a subcellular structure that integrates excitation-contraction coupling and energy metabolism. Here, using this model, we examined the influence of distance between mitochondria and Ca²⁺ release sites by comparing a normal (50-nm) distance model and a large (200-nm) distance model (LD). The influence of distance was minimal under a low pacing rate (0.25 Hz), but under a higher pacing rate (2 Hz), lower levels of mitochondrial Ca²⁺ and NADH, elevated phosphate, and suppressed force generation became apparent in the LD model. Such differences became greater when functional impairments (reduced TCA cycle activity, uncoupling effect, and failing excitation-contraction coupling) were additionally imposed. We concluded that juxtaposition of the mitochondria and the Ca²⁺ release sites is crucial for rapid signal transmission to maintain cardiac-energy balance. The idealized 3D model of cardiac excitation-contraction and metabolism is a powerful tool to study cardiac energetics.     

Ca2+-induced structural change in the Ca2+Mg2+ domain of troponin C detected by crosslinking

Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca²⁺. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca²⁺, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca²⁺, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the α-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the α-carbon distance His-125-Lys-136, do not exceed 14 Å. Comparison with the X-ray structure of troponin C (Herzberg, O, and James, M.N.G. (1985) Nature 313, 653–659) indicates that some of the above distances increase on Ca²⁺-binding.     

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A,Mg2+ Ap5A,and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap₅A, Mn²⁺ Ap₅A, and Mg²⁺ Ap₅A have been determined by X-ray crystallography to resolutions of 1.6 Å, 1.85 Å, and 1.96 Å, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap₅A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn²⁺ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 Å. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg²⁺ Ap₅A and Mn²⁺ Ap₅A structures, Mg²⁺ and Mn²⁺ demonstrate six coordinate octahedral geometry. The interactions of the Mg²⁺-coordinated water molecules with the protein and Ap₅A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for β-γ (preferred by Mg²⁺) rather than α-γ (preferred by Mn²⁺) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation. Proteins 32:276–288, 1998. © 1998 Wiley-Liss, Inc.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

How Does Regulatory Ca2+ Regulate the Na+-Ca2+ Exchanger?

Spatial and temporal regulation of intracellular Ca²⁺ concentrations is a fundamental requirement for life. The mammalian cardiac Na⁺-Ca²⁺ exchanger serves as the main mechanism for Ca²⁺ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca²⁺, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca²⁺. Together with mutational analysis of the Ca²⁺ binding sites, this study reveals the crucial role of one of the two bound Ca²⁺ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.