Second Transmembrane Helix (M2) and Long-range Coupling in Ca2+-ATPase [Bioenergetics]

September 22nd, 2014 by Daiho, T., Yamasaki, K., Danko, S., Suzuki, H.

The Actuator (A) domain of sarco(endo)plasmic reticulum Ca2+-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 Ca2+-transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca2+. 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 Ca2+. 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.