title: Deletions of any single residues in Glu40-Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate.
abstract: Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.
authors.affiliation: Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan.
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: Hajime
authors.initials: H
authors.lastname: Iizuka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics*; D004789:Enzyme Activation; D017353:Gene Deletion; D018698:Glutamic Acid / Q000737:chemistry*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012694:Serine / Q000737:chemistry*
chemical_list: D018698:Glutamic Acid; D012694:Serine; D001216:Asparagine; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D008239:Lysine