Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.
We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca(2+) to the same extent (1-2 Ca(2+) per ATPase monomer) as wild- type ATPase. The inherent ability of the synthetic L6-7 loop peptide to bind Ca(2+) was demonstrated with murexide and mass spectrometry. NMR analysis indicated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum with coordination by all three aspartate residues D813/D815/D818 that resulted in an altered conformation of the peptide chain. Overall our observations suggest that, in addition to mediating contact between the intramembranous Ca(2+) binding sites and the cytosolic phosphorylation site as previously suggested, the L6-7 loop, in a preceding step, participates in the formation of an entrance port important for lodging Ca(2+) at a high-affinity binding site inside the membrane.
Corre
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Section de Biophysique, Departement de Biologie Joliot Curie, CEA et CNRS URA 2096 and LRA17V Université de Paris XI, CE Saclay, 91191 Gif sur Yvette, France.
Jaxel
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Fuentes
J
J
Menguy
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T
Falson
P
P
Levine
B A
BA
Møller
J V
JV
le Maire
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M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium