title: Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites.
abstract: 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.
authors.affiliation: URA 2096 (CNRS) and Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
authors.forename: P
authors.initials: P
authors.lastname: Champeil
authors.forename: T
authors.initials: T
authors.lastname: Menguy
authors.forename: S
authors.initials: S
authors.lastname: Soulié
authors.forename: B
authors.initials: B
authors.lastname: Juul
authors.forename: A G
authors.initials: AG
authors.lastname: de Gracia
authors.forename: F
authors.initials: F
authors.lastname: Rusconi
authors.forename: P
authors.initials: P
authors.lastname: Falson
authors.forename: L
authors.initials: L
authors.lastname: Denoroy
authors.forename: F
authors.initials: F
authors.lastname: Henao
authors.forename: M
authors.initials: M
authors.lastname: le Maire
authors.forename: J V
authors.initials: JV
authors.lastname: Moller
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D008670:Metals / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014462:Ultrafiltration
chemical_list: D008670:Metals; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D000252:Calcium-Transporting ATPases