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.

Second transmembrane helix (M2) and long range coupling in Ca²âº-ATPase.

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.