title: Second transmembrane helix (M2) and long range coupling in Ca²⁺-ATPase.
abstract: The actuator (A) domain of sarco(endo)plasmic reticulum Ca(2+)-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca(2+) transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca(2+). Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P → E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca(2+). Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 → E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis.
authors.affiliation: From the Department of Biochemistry, Asahikawa Medical University, Midorigaoka-Higashi, Asahikawa 078-8510, Japan daiho@asahikawa-med.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Daiho
authors.affiliation: From the Department of Biochemistry, Asahikawa Medical University, Midorigaoka-Higashi, Asahikawa 078-8510, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamasaki
authors.affiliation: From the Department of Biochemistry, Asahikawa Medical University, Midorigaoka-Higashi, Asahikawa 078-8510, Japan.
authors.forename: Stefania
authors.initials: S
authors.lastname: Danko
authors.affiliation: From the Department of Biochemistry, Asahikawa Medical University, Midorigaoka-Higashi, Asahikawa 078-8510, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D000252:Calcium-Transporting ATPases; D002118:Calcium; D005998:Glycine
keywords: Calcium ATPase; Domain Motion; Enzyme Kinetics; Enzyme Mutation; Enzyme Structure; Phosphoenzyme Intermediate; Phosphoryl Transfer; Sarco(endo)plasmic Reticulum; Transmembrane Helix