Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase: Probing nucleotide binding by multidimensional NMR

Mona Abu-Abed, Tapas K. Mal, Masatsune Kainosho, David H. MacLennan, Mitsuhiko Ikura

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca2+ 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.

Original languageEnglish (US)
Pages (from-to)1156-1164
Number of pages9
JournalBiochemistry
Volume41
Issue number4
DOIs
StatePublished - Jan 29 2002

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Reticulum
Calcium-Transporting ATPases
Adenylyl Imidodiphosphate
Nucleotides
Adenosine Triphosphate
Nuclear magnetic resonance
Muscle
Proteolysis
Biomolecular Nuclear Magnetic Resonance
Mutagenesis
Muscle Relaxation
Fluorescein-5-isothiocyanate
Site-Directed Mutagenesis
Titration
Cytosol
Assays
Skeletal Muscle
Crystal structure
Binding Sites
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Abu-Abed, Mona ; Mal, Tapas K. ; Kainosho, Masatsune ; MacLennan, David H. ; Ikura, Mitsuhiko. / Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase : Probing nucleotide binding by multidimensional NMR. In: Biochemistry. 2002 ; Vol. 41, No. 4. pp. 1156-1164.
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Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase : Probing nucleotide binding by multidimensional NMR. / Abu-Abed, Mona; Mal, Tapas K.; Kainosho, Masatsune; MacLennan, David H.; Ikura, Mitsuhiko.

In: Biochemistry, Vol. 41, No. 4, 29.01.2002, p. 1156-1164.

Research output: Contribution to journalArticle

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