Characterization of the ATP-Binding Domain of the Sarco(endo)plasmic Reticulum Ca2+-ATPase: Probing Nucleotide Binding by Multidimensional NMR

详细信息    查看全文

• 作者：Mona Abu-Abed ; Tapas K. Mal ; Masatsune Kainosho ; David H. MacLennan ; and Mitsuhiko Ikura
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：January 29, 2002
• 年：2002
• 卷：41
• 期：4
• 页码：1156 - 1164
• 全文大小：272K
• 年卷期：v.41,no.4(January 29, 2002)
• ISSN：1520-4995

文摘

The skeletal muscle sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1a) mediates musclerelaxation by pumping Ca²⁺ from the cytosol to the ER/SR lumen. In efforts aimed at understanding thestructural 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 byNMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing thenucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity wasdemonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonanceassignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, wasobtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments withAMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket regionidentified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directedmutagenesis studies of SERCA1a.