title: Roles of interaction between actuator and nucleotide binding domains of sarco(endo)plasmic reticulum Ca(2+)-ATPase as revealed by single and swap mutational analyses of serine 186 and glutamate 439.
abstract: Roles of hydrogen bonding interaction between Ser(186) of the actuator (A) domain and Glu(439) of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca(2+)-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca(2+)-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wild-type rates. Results showed that the Ser(186)-Glu(439) hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm approximately mm range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser(186) and Glu(439) are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.
authors.affiliation: Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Liu
authors.forename: Takashi
authors.initials: T
authors.lastname: Daiho
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Guoli
authors.initials: G
authors.lastname: Wang
authors.forename: Stefania
authors.initials: S
authors.lastname: Danko
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006860:Hydrogen Bonding; D008954:Models, Biological; D008968:Molecular Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000235:genetics
chemical_list: D018698:Glutamic Acid; D012694:Serine; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases