title: Characterization of sarcoplasmic reticulum Ca2+ ATPase nucleotide binding domain mutants using NMR spectroscopy.
abstract: Sarcoplasmic reticulum Ca(2+) ATPase (SERCA) is essential for muscle function by transporting Ca(2+) from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolated SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. (15)N-(1)H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant (∼2.5 mM) was similar to that of WT (∼3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Wazo
authors.initials: W
authors.lastname: Myint
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Gong
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Ahn
authors.forename: Rieko
authors.initials: R
authors.lastname: Ishima
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics
chemical_list: D000249:Adenosine Monophosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases