Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction.
Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction.
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通过脂质-蛋白质相互作用研究质膜钙泵亚型 2 和 4 的自抑制机制。
DOI：
10.1042/bj20111035
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Rossi,JuanPabloFC
中科院分区：
文献类型：
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作者：
Mangialavori,IreneC;Corradi,Gerardo;Rinaldi,DeboraE;delaFuente,MariaCandelaria;Adamo,HugoP;Rossi,JuanPabloFC
PMCA（质膜Ca 2 +-ATP酶）的自抑制/激活涉及蛋白质的膜区域的构象变化，其影响与跨膜结构域直接相关的脂质的量。使用磷脂酰胆碱类似物[125 I]TID-PC/16 {1-O-十六烷酰基-2-O-[9-[[[2-[125 I]碘-4-基]-N-甲基-N-（2-甲基-N-甲基-N-苯基）-N-甲基-N-（2-甲基-N-甲基-N-苯基）-N-（2-甲基-N-甲基-N-苯基）-N-（2-甲基-N-苯基）-N-（2-甲基-N-苯基）-N-（2-甲基-N-（2-甲基-N-苯基）-N-（2-甲基-N-苯基）-N-（（三氟甲基-3H-二氮杂环丙烯-3-基）苄基]氧基]羰基]壬酰基]-sn-甘油基-3-磷酸胆碱}，将其掺入二肉豆蔻酰磷脂酰胆碱和非离子去污剂C12 E10 [十（乙二醇）十二烷基醚]的混合物中。我们发现重组PMCA 4和从红细胞中纯化的PMCA（ePMCA）之间没有差异。然而，通过PMCA 2的Ca 2 +/钙调蛋白的半最大激活的滴定显示出比PMCA 4高30倍的亲和力。PMCA 2表现出较低水平的标记在自抑制构象相对于PMCA 4，表明较低的自抑制与较低的暴露于脂质的自抑制状态。脂质-蛋白质化学计量的分析表明，PMCA的脂质环变化：（i）根据酶的构象状态;和（ii）取决于不同的异构体的PMCA。PMCA 2在Ca 2+转运过程中改变其构象的程度比PMCA 4小，PMCA 4是一种对钙调蛋白和酸性磷脂的调节更敏感的同种型。这是第一次演示的环状脂质和PMCA的动态行为。
The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid–protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form fromSaccharomyces cerevisiaewas investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10[deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid–protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.