Localization of a putative magnesium-binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca(2+)-ATPase.
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 Ca(2+)-ATPase (SERCA-1), this region is located at residues 700-712. 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-cation-binding sites. A 38-residue polypeptide, corresponding to the domain 682-719 of the Ca(2+)-ATPase was synthesized and tested for its ability to bind divalent cations. Circular-dichroism, intrinsic-fluorescence and fluorescence-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 Mg2+, 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 alpha-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 Mg2+ in the nucleotide-binding site and/or in the phosphorylation site of P-type ATPases.
Girardet
J L
JL
Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Bally
I
I
Arlaud
G
G
Dupont
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent; D002942:Circular Dichroism; D003593:Cytoplasm / Q000201:enzymology; D004735:Energy Transfer; D005453:Fluorescence; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013725:Terbium / Q000378:metabolism; D014270:Trifluoroethanol / Q000494:pharmacology
D002413:Cations, Divalent; D010446:Peptide Fragments; D013725:Terbium; D014270:Trifluoroethanol; D000252:Calcium-Transporting ATPases; D008274:Magnesium