Critical role of glu40-ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme
Critical role of glu40-ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme
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DOI：
10.1074/jbc.m707665200
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发表时间：
2007-11-23
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
通过在Pro(42)/Ala（43）和Gly(46)/Lys（47）处插入甘氨酸，探讨了连接执行器结构域和肌浆网Ca2+- atp酶的第一跨膜螺旋的A/M1连接器(Glu（40)-Ser(48)）长度的功能重要性。在每个位点插入两个或更多的甘氨酸完全消除atp酶的活性。在这些突变体中，磷酸酶（EP）中间体从adp敏感型（E1P）到adp不敏感型（E2P）的异构化明显加快，但EP的衰变被完全阻断。因此，积累的E2P被证明是E2PCa(2)，在运输位点具有两个闭塞的Ca2+离子，Ca2+的解封和释放在突变体中被阻断。相比之下，由无Ca2+的Pi产生的无Ca2+形式的E2P的水解在突变体中与野生型一样快。对胰蛋白酶和蛋白酶K的抗性分析表明，积累的E2PCa(2)结构介于E1PCa(2)和Ca2+释放的E2P状态之间。即在E2PCa(2)中，促动器结构域已经从其在E1PCa(2)中的位置大量旋转，并与Ca2+释放的E2P状态中的磷酸化结构域相关联；然而，在E2PCa(2)中，这些结构域与第二跨膜螺旋顶部的Leu(119)/Tyr（122）之间的疏水相互作用尚未形成。这与我们之前的发现一致，即Tyr（122）上的这些相互作用对于Ca2+释放的E2P结构的形成至关重要。结果表明，EP异构化/Ca2+释放过程包括两个步骤：E1PCa(2) -> E2PCa(2) -> E2P + 2Ca(2+)；首次鉴定到中间态E2PCa(2)。结果进一步表明，A/M1连接体长度较短，可能是由于E2PCa中施加的应变(2)，它对于致动器和磷酸化结构域的正确定位和相互作用至关重要，从而导致Ca2+解除和释放的结构变化。
The functional importance of the length of the A/M1 linker (Glu(40)-Ser(48)) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca2+-ATPase was explored by its elongation with glycine insertion at Pro(42)/Ala(43) and Gly(46)/Lys(47). Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa(2) possessing two occluded Ca2+ ions at the transport sites, and the Ca2+ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca2+-free form of E2P produced from Pi without Ca2+ was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa(2) accumulated is an intermediate state between E1PCa(2) and the Ca2+-released E2P state. Namely in E2PCa(2), the actuator domain is already largely rotated from its position in E1PCa(2) and associated with the phosphorylation domain as in the Ca2+-released E2P state; however, in E2PCa(2), the hydrophobic interactions among these domains and Leu(119)/Tyr(122) on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr(122) are critical for formation of the Ca2+-released E2P structure. Results showed that the EP isomerization/Ca2+-release process consists of the following two steps: E1PCa(2) -> E2PCa(2) -> E2P + 2Ca(2+); and the intermediate state E2PCa(2) was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa(2), is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca2+ deocclusion and release.