Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase
Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase
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DOI：
10.1074/jbc.m116.759704
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发表时间：
2016-11-18
影响因子：
4.8
通讯作者：
Suzuki, Hiroshi
中科院分区：
文献类型：
--
作者：
Daiho, Takashi;Yamasaki, Kazuo;Suzuki, Hiroshi
sarco（endo）质网Ca2+- atp酶的细胞质致动器结构域经历大的旋转运动，影响远的跨膜运输位点，与该结构域连接的长第二跨膜螺旋（M2）在细胞质催化结构域和运输位点之间的传递运动中起关键作用。在这里，我们通过引入限制/增加构象自由的突变，探索了Gly（105）在M2的细胞质（M2c）和跨膜（M2m）段之间可能的结构作用。丙氨酸取代G105A显着延缓了磷酸酶中间体(E1PCa(2) -> E2PCa（2) -> E2P + 2Ca(2+)）的异构化，并破坏了E1PCa(2)和E2PCa(2)在转运位点的Ca2+闭塞，使ATP水解和Ca2+转运不耦合。相反，这种取代加速了atp酶的激活（E2 -> E1Ca(2)）。通过在G105A突变体M2上替换另一个残基引入甘氨酸(即。“g移位取代”)确定每个步骤中适当的Ca2+处理和动力学所需的甘氨酸位置。所有野生型的动力学性质，包括耦合输运，都完全恢复了位于M2c螺旋同侧的位置112（G105A/A112(4)）与Gly（105）面对M4/磷酸化结构域。结果表明，Gly（105）在Ca2+转运周期中起着柔性膝关节的作用，因此细胞质结构域运动可以使M2向正确方向弯曲和应变，或使螺旋拉直，以适当地调节和耦合Ca2+转运和ATP水解。
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 Gly(105) 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 (E1PCa(2) -> E2PCa(2) -> E2P + 2Ca(2+)), and disrupts Ca2+ occlusion in E1PCa(2) and E2PCa(2) at the transport sites uncoupling ATP hydrolysis and Ca2+ transport. In contrast, this substitution accelerates the ATPase activation (E2 -> E1Ca(2)). 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 (4-shift substitution at position 112 (G105A/A112(4) located on the same side of the M2c helix as Gly(105) facing M4/phosphorylation domain. Results demonstrate that Gly(105) 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.