Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
PLoS One. 2012;7(7):e40369. doi: 10.1371/journal.pone.0040369. Epub 2012 Jul 11.
The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.
肌浆网/内质网钙 ATP 酶（SERCA）在转运钙的过程中发生构象变化，但结构域运动的细节仍不清楚。本研究的目的是测量 SERCA2a 细胞质结构域之间的距离，以揭示构象变化的幅度和方向。通过活细胞荧光显微镜，我们测量了融合到 SERCA N 端的供体荧光蛋白与融合到 N 端、P 端或跨膜结构域的受体荧光蛋白之间的分子内荧光共振能量转移（FRET）。体外 ATP 酶活性和活细胞中 Ca 摄取表明“双荧光蛋白”SERCA 构建体具有催化活性。所有构建体均表现出对泵配体钙和 thapsigargin（Tg）的动态 FRET 变化。这些 FRET 变化被量化为 SERCA 构象变化的指标。与 Tg 结合时，两个 N 结构域融合位点（残基 509 或 576）的分子内 FRET 减少，而 P 结构域（残基 661）和 TM 结构域（C 端）融合则显示出与 Tg 结合时 FRET 增加。磷蛋白（PLB）的共表达并未降低 Tg 依赖性构象变化的幅度，PLB 也没有减缓 Tg 结合的动力学。与所有构建体相比，离子载体通透细胞中的 FRET 在 EGTA 中低于在饱和钙中，这表明与从 E2（无钙）到 E1（结合钙）的结构转变相关的结构域分离距离减小。这些数据表明钙结合后细胞质头盖关闭。本研究结果提供了对 Ca-ATP 酶结构动力学的深入了解。此外，本研究开发的双荧光蛋白 SERCA 构建体可能有助于评估候选小分子对 Ca 摄取活性的调节剂。
