Conformational Changes of SERCA Revealed by Intramolecular FRET

Abstract

The first structures of the sarcoendoplasmic reticulum ATPase (SERCA) determined by X-ray crystallography suggested that pump undergoes a large conformational change during catalytic cycling. The transition from the E1 (Ca-bound) state to the E2 (Ca-free) state was predicted to decrease inter-domain separation distance with closure of the SERCA cytoplasmic headpiece. To test this model, we fused Cerulean to the A-domain and YFP to the N or P or TM-domain of SERCA2a. These “2-color” SERCA constructs were expressed in AAV cells, and SERCA structure transitions were detected by changes in intramolecular fluorescence resonance energy transfer (FRET). FRET decreased with thapsigargin for the two N-domain fusion sites (residues 510, 577), while the P- (residue 610) and TM- domain (C-terminus) fusions showed increased FRET with thapsigargin. Unexpectedly, FRET in permeabilized cells was higher in Ca [10 μM] than in EGTA for all constructs, suggesting an increase in domain separation distance with the E1 to E2 transition. These observations were supported by parallel experiments in fluorescence lifetime distribution (FLD) analysis. FLD resolved two broad distributions of fluorescence lifetimes for 2-color SERCA expressed in live cardiac myocytes, consistent with two major FRET states. The relative populations of these states oscillated with electrical pacing, favoring the high FRET (short distance) state during systole (contraction) and the low FRET (long distance) state during diastole (relaxation). We expect 2-color SERCA constructs to be useful for exploring the magnitude, direction, and kinetics of calcium pump conformational changes. They may also be useful for screening candidate compounds for modulation of SERCA pump structure and function.