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J Biol Chem, Vol. 273, Issue 12, 6619-6631, March 20, 1998

Characterization of a Protease-resistant Domain of the Cytosolic Portion of Sarcoplasmic Reticulum Ca²⁺-ATPase
NUCLEOTIDE- AND METAL-BINDING SITES

Philippe Champeil, Thierry Menguy, Stéphanie Soulié, Birte Juul, Adrienne Gomez de Gracia, Filippo Rusconi^**, Pierre Falson, Luc Denoroy^¶¶, Fernando Henao^||, Marc le Maire, and Jesper Vuust Møller

From the  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, ^** Laboratoire de Neurobiologie et Diversité Cellulaire, CNRS (URA 2054) and Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris, 10 rue Vauquelin, 75231 Paris Cedex 05, ^¶¶ Service Central d'Analyse (CNRS), BP 22, 69390 Vernaison, France, ^|| Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, 06080 Badajoz, Spain, and  Department of Biophysics, University of Aarhus, Ole Worms Allé 185, 8000 Aarhus C, Denmark

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.

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