Second Transmembrane Helix (M2) and Long Range Coupling in Ca2+-ATPase

Takashi Daiho,Kazuo Yamasaki,Stefania Danko,Hiroshi Suzuki

doi:10.1074/jbc.m114.584086

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.

Highlights

The catalytic A domain of Ca2ϩ-ATPase moves substantially and connects to distant Ca2ϩ sites through transmembrane helices M1 and M2
Our results demonstrate that different parts of the A/M2 link play a critical role in synchronizing gating of the two Ca2ϩ at the transport sites on both sides of the membrane, and each is part of the mechanism for coupling catalytic and transport site structural events
ATP Hydrolysis, Ca2ϩ Transport, and Their Coupling—First, we explored possible effects of 5Gi mutations on the overall transport cycle

Summary

Background

The catalytic A domain of Ca2ϩ-ATPase moves substantially and connects to distant Ca2ϩ sites through transmembrane helices M1 and M2. The actuator (A) domain of sarco(endo)plasmic reticulum Ca2؉-ATPase plays a catalytic role and undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. We focus on each region of the long helix M2: transmembrane M2m, cytoplasmic M2c, M2top, and A/M2junction (Fig. 1B) We explored their roles and the functional significance of the changes in secondary structure and length for the coupling of the different conformational steps that are required to efficiently convert the chemical energy of ATP hydrolysis into the changes in accessibility, orientation, and affinity of the Ca2ϩ binding transport sites. Our results demonstrate that different parts of the A/M2 link play a critical role in synchronizing gating of the two Ca2ϩ at the transport sites on both sides of the membrane, and each is part of the mechanism for coupling catalytic and transport site structural events. M2 and its connection with the A domain have a role distinct from that of the A/M1Јlinker loop [23,24,25], both are needed to coordinate coupling

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION