Instituto de Química y Fisicoquímica Biologicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Junín 956, 1113 Buenos Aires, Argentina.
J Biol Chem. 2011 May 27;286(21):18397-404. doi: 10.1074/jbc.M110.210088. Epub 2011 Mar 31.
The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.
质膜钙泵（PMCA）暴露于周围磷脂的情况通过测量光活化磷脂酰胆碱类似物[(125)I]TID-PC/16与蛋白的结合来评估。在 Ca(2+)存在的情况下，钙调蛋白（CaM）和磷脂酸（PA）都极大地降低了[(125)I]TID-PC/16与 PMCA 的结合。用 V8 蛋白酶对 PMCA 进行蛋白水解会产生三个主要片段：N，包括跨膜片段 M1 和 M2；M，包括 M3 和 M4；C，包括 M5 到 M10。CaM 降低了[(125)I]TID-PC/16与片段 M 和 C 的结合水平，而磷脂酸降低了[(125)I]TID-PC/16与片段 N 和 M 的结合水平。这表明结合 CaM 或 PA 引起的构象变化延伸到相邻的跨膜结构域。有趣的是，这一结果还表示 CaM 和 PA 产生的活性构象之间存在差异。为了验证这一点，我们测量了 PMCA 在 ATP 结合位点用异硫氰酸酯标记的曙红与 PMCA 胶束中包含的磷脂 RhoPE 之间的共振能量转移。CaM 降低了这两个探针之间能量转移的效率，而 PA 则没有。这一结果表明，CaM 的激活增加了 ATP 结合位点与膜之间的距离，但 PA 不影响这个距离。我们的结果揭示了 CaM 或 PA 诱导的 PMCA 构象之间的主要差异，并表明这些差异涉及跨膜区域。
