title: Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca(2+)-ATPase: probing nucleotide binding by multidimensional NMR.
abstract: The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.
authors.forename: Mona
authors.initials: M
authors.lastname: Abu-Abed
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Mal
authors.forename: Masatsune
authors.initials: M
authors.lastname: Kainosho
authors.forename: David H
authors.initials: DH
authors.lastname: MacLennan
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D017386:Sequence Homology, Amino Acid
chemical_list: D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases