title: Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism.
abstract: We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).
authors.affiliation: Unité de Recherche Associée 2096 of the Centre National de la Recherche Scientifique, Département de Biologie Joliot Curie, CEA Saclay, 91191 Gif-sur-Yvette Cedex, Université Paris Sud, France.
authors.forename: Cedric
authors.initials: C
authors.lastname: Montigny
authors.forename: Christine
authors.initials: C
authors.lastname: Jaxel
authors.forename: Alla
authors.initials: A
authors.lastname: Shainskaya
authors.forename: Joëlle
authors.initials: J
authors.lastname: Vinh
authors.forename: Valérie
authors.initials: V
authors.lastname: Labas
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Møller
authors.forename: Steven J D
authors.initials: SJ
authors.lastname: Karlish
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D005296:Ferrous Compounds / Q000494:pharmacology*; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
chemical_list: D005296:Ferrous Compounds; D010446:Peptide Fragments; D000266:Adenylyl Imidodiphosphate; D000252:Calcium-Transporting ATPases