2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding.
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.
Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
Zhanjia
Z
Hou
Zhihong
Z
Hu
Daniel J
DJ
Blackwell
Tyler D
TD
Miller
David D
DD
Thomas
Seth L
SL
Robia
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003116:Color; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D000229:Dependovirus / Q000378:metabolism; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008856:Microscopy, Fluorescence; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium