title: Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase.
abstract: The cytoplasmic actuator domain of the sarco(endo)plasmic reticulum Ca2+-ATPase undergoes large rotational movements that influence the distant transmembrane transport sites, and a long second transmembrane helix (M2) connected with this domain plays critical roles in transmitting motions between the cytoplasmic catalytic domains and transport sites. Here we explore possible structural roles of Gly105between the cytoplasmic (M2c) and transmembrane (M2m) segments of M2 by introducing mutations that limit/increase conformational freedom. Alanine substitution G105A markedly retards isomerization of the phosphoenzyme intermediate (E1PCa2→ E2PCa2→ E2P + 2Ca2+), and disrupts Ca2+occlusion in E1PCa2and E2PCa2at the transport sites uncoupling ATP hydrolysis and Ca2+transport. In contrast, this substitution accelerates the ATPase activation (E2 → E1Ca2). Introducing a glycine by substituting another residue on M2 in the G105A mutant (i.e. "G-shift substitution") identifies the glycine positions required for proper Ca2+handling and kinetics in each step. All wild-type kinetic properties, including coupled transport, are fully restored in the G-shift substitution at position 112 (G105A/A112G) located on the same side of the M2c helix as Gly105facing M4/phosphorylation domain. Results demonstrate that Gly105functions as a flexible knee-like joint during the Ca2+transport cycle, so that cytoplasmic domain motions can bend and strain M2 in the correct direction or straighten the helix for proper gating and coupling of Ca2+transport and ATP hydrolysis.
authors.affiliation: From the Department of Biochemistry, Asahikawa Medical University, Midorigaoka-Higashi, Asahikawa 078-8510, Japan. Electronic address: 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: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D020125:Mutation, Missense; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium; D005998:Glycine
keywords: calcium ATPase; domain motion; enzyme kinetics; enzyme mechanism; enzyme mutation; enzyme structure; glycine; phosphoenzyme intermediate; sarco(endo)plasmic reticulum; transmembrane helix