Abstract
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.
Highlights
The sarco(endo)plasmic reticulum calcium adenosine triphosphatase (SERCA) is the ion-motive ATPase responsible for maintaining the 7,000 fold Ca gradient [1] across the membrane of the endoplasmic reticulum
This suggests a decrease in the distance between the Cer donor fluorophore and the YFP acceptor, which reveals that the SERCA cytoplasmic headpiece becomes more compact after Ca binds to the SERCA transmembrane domain
This change was somewhat smaller in magnitude than the 30+ Achange predicted by the first X-ray crystallographic structures (Fig. 1A), but larger than that measured by other fluorescence resonance energy transfer (FRET) studies [8,9,29], Most notably, the distance change observed here was opposite in direction compared to early crystal structure predictions [4]
Summary
The sarco(endo)plasmic reticulum calcium adenosine triphosphatase (SERCA) is the ion-motive ATPase responsible for maintaining the 7,000 fold Ca gradient [1] across the membrane of the endoplasmic reticulum. X-ray crystallography has provided many atomic resolution SERCA structures suggesting that the relative motions of SERCA cytoplasmic domains result in opening and closing of the cytoplasmic headpiece during the transition between the E1 (Ca-bound) and E2 (Ca-free) enzymatic substate (Fig. 1). Many aspects of these crystallographic results have been confirmed by other methods but some details of the domain motions are still unclear. Later crystal structures, determined in the presence of nucleotide analogs, suggest that the headpiece remains closed throughout the catalytic cycle.
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