Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase.

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 Gly¹⁰⁵ between 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 E1PCa₂ and E2PCa₂ at 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 Gly¹⁰⁵ facing M4/phosphorylation domain. Results demonstrate that Gly¹⁰⁵ functions 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.

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