Evidence that the ATP binding site of sarcoplasmic reticulum CaATPase has a Mg(2+) ion binding sub-site

doi:10.1016/0006-291X(84)90934-3

Biochemical and Biophysical Research Communications

Volume 124, Issue 1, 15 October 1984, Pages 183-189

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Abstract

The CaATPase of skeletal muscle sarcoplasmic reticulum was specifically labeled in the ATP binding site with fluorescein isothiocyanate under gentle conditions (pH 7·5). Fluorescence energy transfer from the attached fluorescein to Nd³⁺ indicated that a cation binding site was about 1·0 nm away from the fluorescein. Thus it appears that the ATP site includes a cation binding site. At 25°C in 0·5 M KCl, the association constants for Nd³⁺, Ca²⁺ and Mg²⁺ were 3·3×10⁵ M⁻¹, 84 M⁻¹ and 35 M⁻¹, respectively, making it possible that, $in vivo$ , the site binds Mg²⁺.

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Cited by (30)

Interactions of phosphate groups of ATP and aspartyl phosphate with the sarcoplasmic reticulum Ca2+-ATPase: An FTIR study
2005, Biophysical Journal
Citation Excerpt :
However, if the photolysis spectrum of the reaction 2 caged ATP → 2 free ATP is subtracted, the absorption of 1 free ATP appears negative in the resulting binding spectrum.) No significant absorbance changes were observed in control samples that contained the inhibitor thapsigargin (16), which selectively inhibits the Ca2+-ATPase (45–47); 20 mM EGTA (48), which converts the ATPase to a Ca2+-free state; or fluorescein isothiocyanate-labeled ATPase (48), which blocks the ATP binding site (49,50). We checked the robustness of our subtraction procedures in several ways:
Phosphate binding to the sarcoplasmic reticulum Ca²⁺-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([β-¹⁸O₂, βγ-¹⁸O]ATP and [γ-¹⁸O₃]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm⁻¹ are affected by labeling of the β-phosphate, bands likely near 1154, and 1098–1089 cm⁻¹ are affected by γ-phosphate labeling. The findings indicate that the strength of interactions of β- and γ- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm⁻¹, were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca₂E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca₂E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca₂E1P phosphate less susceptible to attack by water.
Preparation of Na,K-ATPase specifically modified on the anti-fluorescein antibody-inaccessible site by fluorescein 5'-isothiocyanate
2000, Analytical Biochemistry
Specific labeling is required for energy transfer measurements and to avoid artifacts in the use of fluorophores as reporter groups. Therefore, a method for specific modification by one of the most popular reagents for P-type ATPases (fluorescein 5′-isothiocyanate) has been developed. Sulfhydryl reagents protected against modification of cysteine residues, and treatment with dithiothreitol eliminated a slow doubling of the fluorescence of conventionally modified Na,K-ATPase upon dilution that is attributed to disappearance of self-energy transfer. Removal of nonspecifically bound fluorescein was also confirmed by titration of the modified Na,K-ATPase with anti-fluorescein antibody and by time resolution of the fluorescence change when the modified enzyme was mixed with Na⁺ in a stopped-flow instrument. The only fluorescence change when specifically modified Na,K-ATPase was mixed with Na⁺ was the signal from fluorescein at the antibody-inaccessible, substrate-protectable site that reports the conformational change in unphosphorylated enzyme. The magnitude of the fluorescence change reporting the conformational change increased from between 8 and 12% to between 25 and 30% without affecting the kinetic constants estimated from titrations with Na⁺ and K⁺. The method should be generally applicable to the preparation of specifically labeled P-type pumps for use in kinetic and equilibrium titrations or energy transfer measurements.
Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites
1998, Journal of Biological Chemistry
Treatment of rabbit sarcoplasmic reticulum Ca²⁺-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp³⁵¹ and ending in the Lys⁶⁰⁵-Arg⁶¹⁵ region, well before the conserved sequences generally described as constituting the “hinge” region of this P-type ATPase (residues 670–760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca²⁺-ATPase as follows: high sensitivity of peptidic bond Arg⁵⁰⁵-Ala⁵⁰⁶ to trypsin cleavage, and high reactivity of lysine residue Lys⁵¹⁵ toward the fluorescent label fluorescein 5′-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca²⁺-ATPase. The fragments also bind Nd³⁺ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670–760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.
Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca2+-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site
1993, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
The sarcoplasmic reticulum Ca²⁺-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg²⁺ and absence of Ca²⁺. This inactivation was Mg²⁺-dependent, and prevented by high-affinity Ca²⁺ binding. The enzyme was completely protected by ATP against the inactivation with an affinity consistent with that of the catalytic site for ATP. The affinity for Mg²⁺ in this inactivation was in agreement with that for Mg²⁺ in phosphorylation of the enzyme with P_i. Mg · ATP did not bind to the inactivated enzyme, whereas metal-free ATP did bind to it with a high affinity. These findings suggest that the Mg²⁺ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg²⁺. The enzyme was completely protected by P_i against the inactivation with an affinity consistent with that of the catalytic site for P_i. The inactivated enzyme showed neither phosphorylation with P_i nor high-affinity vanadate binding. These findings suggest that the phosphorylation site of the inactivated enzyme is occupied by tightly-bound F^-. The contents of tightly-bound Mg²⁺ and F^- in the inactivated enzyme were determined after unbound Mg²⁺ and F^- were removed by gel filtration. 2.3 mol of Mg²+ and 3.7 mol of F^- per mol of phosphorylation sites were tightly bound to the enzyme. The tight binding of these ligands depended on the presence of each other, and was completely prevented by high-affinity Ca²⁺ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg²⁺ and F^- were entirely released by low-affinity Ca²⁺ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg²⁺ and F^- to the catalytic site.
Effects of pH, Ca2+ and lanthanides on conformation of the sarcoplasmic reticulum Ca2+-ATPase catalytic site
1992, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
The conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC) bound to the adenine subsite. The FITC-SR fluorescence parameters have been examined in the pH range 5.7–8.0 in the presence of EGTA, Ca²⁺ or Ln³⁺ (La³⁺, Pr³⁺, Nd³⁺, Tb³⁺ etc.). A quantitative method to calculate the equilibrium between the protein conformers is proposed on the basis of the fluorometric titration curve analysis. The distance Nd³⁺-FITC was estimated to be about 1 nm at pH 6–7 and 1.7 nm at pH 8 which can be interpreted as an increase of the distance between the nucleotide and phosphorylation domains of Ca²⁺-ATPase in alkaline media. These studies suggest that the ligand-stabilized E₁-form of Ca²⁺-ATPase can exist in two conformational states with the closed and opened interdomain cleft in the pH range 5.7–8.0. The pH-dependence of the ratio of these states correlates with that of the E₁ ↔ E₂ equilibrium without ligands. These dependences were approximated by simple Henderson-Hassellbach equations with pK 7.0 ± 0.1, i.e. the transition between two protein conformations is probably governed by one proton dissociation.
The Ca2+ transport ATPases of sarco(endo)-plasmic reticulum and plasma membranes
1992, New Comprehensive Biochemistry
The Ca²⁺ transport ATPase of sarcoplasmic reticulum is an intrinsic membrane protein of 110 kDa that controls the distribution of intracellular Ca²⁺ by ATPdependent translocation of Ca²⁺ ions from the cytoplasm into the lumen of the sarcoplasmic reticulum. This chapter discusses recent developments made on the structure of the Ca²⁺-ATPase of the sarcoplasmic reticulum, in addition to the Ca²⁺-ATPases in the plasma membranes and endoplasmic reticulum of non-muscle cells. The chapter focuses on the various forms of the Ca²⁺ transport ATPases that occur in the sarcoplasmic reticulum of the muscle cells of diverse fiber types and in the endoplasmic reticulum of nonmuscle cells (SERCA). It also compares the structure of these enzymes are compared with the Ca²⁺ transport ATPases of surface membranes (PMCA) and with other ATP-dependent ion pumps that transport Na⁺, K⁺, and H⁺. The sarco(endo)plasmic reticulum Ca²⁺-ATPases of mammalian tissues can be divided structurally into three main groups (SERCA 1–3) representing the products of different genes.

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Biochemical and Biophysical Research Communications

Abstract

References (19)

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biophys. Biochim. Acta

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J. Biol. Chem

Cited by (30)

Interactions of phosphate groups of ATP and aspartyl phosphate with the sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase: An FTIR study

Preparation of Na,K-ATPase specifically modified on the anti-fluorescein antibody-inaccessible site by fluorescein 5'-isothiocyanate

Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase. Nucleotide- and metal-binding sites

Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site

Effects of pH, Ca<sup>2+</sup> and lanthanides on conformation of the sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase catalytic site

The Ca<sup>2+</sup> transport ATPases of sarco(endo)-plasmic reticulum and plasma membranes