Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca²⁺‐ATPase

Abstract

The amino acid sequences of several P-type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the 'hinge' domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca2+-ATPase (SERCA-l), this region is located at residues 700-71 2.Comparison of the sequence in this domain with calcium-binding proteins reveals similarities with the center of the helix-loop-helix EF-hand structure that is known to form divalent-cationbinding sites. A 38-residue polypeptide, corresponding to the domain 682-719 of the Ca'+-ATPase was synthesized and tested for its ability to bind divalent cations.Circular-dichroism, intrinsic-fluorescence and tluorescence-energy-transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg", with an affinity of 10-15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the a-helix promoter 2,2,2-trifluoroethanol.Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties.It is proposed that this sequence is involved in the co-ordination of a Mgz+ in the nucleotidebinding site and/or in the phosphorylation site of P-type ATPases.The sarcoplasmic reticulum Ca2+-ATPase is a member of the P-type ATPases family. It is proposed that the Ca2'-ATPase is composed of 10 transmembrane a-helices and a cytoplasmic globular fraction that is divided into two main domains, a small and a large cytoplasmic loop. The large cytoplasmic loop (amino acid residues 330-740) contains the phosphorylation site (Asp351), and most probably the ATPbinding and phosphate-binding sites (McLennan et al., 1985).Several regions of the large loop show a high degree of similarity within P-type ATPases. One of these regions, located around residues 696-707 in the CaZ+-ATPase, the socalled 'hinge' region, presents the highest degree of conservation among the sequences of P-type ATPases (Serrano, 1988) It has been shown that mutations of Thr701, Gly702, Asp703 or Asp707 strongly reduce or totally inhibit the calcium-transport activity of the Ca'+-ATPase (Clarke et al., 1990). The corresponding sequence in NdK-ATPase can be labeled by 5'-(p-fluorosulfonyl)benzoyladenosine, an ATP analogue that inhibits the ATPase activity of this enzyme (Ohta et al., 1986).These observations indicate that the integrity of this part of the protein is essential for the activity of the P-type AT- Pases, and suggest that it may be involved in the binding of ATP to the active site.It has been proposed that the area surrounding the Asp638 of a yeast proton-pumping ATPase (Serrano, 1988;Portillo and Serrano, 1988) could be involved in the binding of the y phosphate of ATP through a chelate...

NMR structures of the C-terminal end of human complement serine protease C1s

“…Deprotection and cleavage of the peptides from the resin was performed with trifluoromethane sulphonic acid and removal of the protecting formyl group from tryptophan was achieved by treatment of the peptide for 5 min at 0°C with 1 M ethanolamine in 6 M guanidine-HCl, pH 11. Reduction of methionine sulphoxide generated during synthesis was achieved by treatment of the peptide with N-methylmercaptoacetamide as described previously [7]. The peptides were purified by preparative reverse-phase high-pressure liquid chromatography (HPLC) on a 30-nm Vydac C18 column (2.2 cm ×25 cm, 10 mm) using a 30-min linear gradient of acetonitrile (5 -60%) in 0.1% trifluoroacetic acid.…”

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

“…Analogous results were obtained for the homologous residues in CaATPase (10). A role in Mg 2ϩ binding was proposed on the basis of peptide binding studies (11) and because mutation of Asp 710 in the ␣-subunit of renal Na,K-ATPase did not interfere with free ATP binding (12). Contributions of Asp 710 and Asp 714 to coordination of Mg 2ϩ was also proposed in attempts at modeling the active site of cation ATPases on the dehalogenase fold (13).…”

A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorganic phosphate

“…SERCA activity is highly dependent on the availability of both Mg 2+ and ATP [23]. Figure 1 depicts the dependence of 45 Ca 2+ accumulation in microsomes on increasing concentrations of Mg 2+ .…”