Interdomain Fluorescence Resonance Energy Transfer in SERCA Probed by Cyan-Fluorescent Protein Fused to the Actuator Domain

Abstract

We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in the N domain. At low [Ca (2+)] (E2 biochemical state), the measured FRET efficiency between CFP (donor in A domain) and FITC (acceptor in N domain) was 0.34 +/- 0.03, indicating a mean distance of 61.6 +/- 2.0 A between probes on the two domains. An increase of [Ca (2+)] to 0.1 mM (E1-Ca biochemical state) decreased the FRET efficiency by 0.06 +/- 0.03, indicating an increase in the mean distance by 3.0 +/- 1.2 A. Quantitative molecular modeling of dual-labeled SERCA, including an accurate calculation of the orientation factor, shows that the FRET data observed in the absence of Ca is consistent with the E2 crystal structure, but the increase in distance (decrease in FRET) induced by Ca is much less than predicted by the E1 crystal structure. We conclude that the E1 crystal structure does not reflect the predominant structure of SERCA under physiological conditions in a functional membrane bilayer.

Oligomeric Interactions of Sarcolipin and the Ca-ATPase

“…cDNA encoding rabbit SERCA1a (Enzyme Collection Number 03.06.03.08) was provided by Dr. David MacLennan (University of Toronto). cDNAs encoding CFP and YFP were purchased from Clontech (37). DNA mutagenesis QuikChange TM kit was purchased from Stratagene.…”

“…S1). cDNA encoding CFP or YFP was fused to the initiator methionine codon of SLN cDNA using a restriction enzyme/ligation strategy (Nco1/Sac1) that inserted three amino acids (glycine-glutamate-leucine) (37). Residue alanine-206 of CFP and YFP was mutated to lysine (A206K) to disrupt dimerization by GFP and its mutant derivatives (39).…”

“…Molecular Modeling-The molecular model of CFPSERCA1a in the calcium-free state was constructed using DS Visualizer (Accelrys, San Diego, CA) and Fusion Protein Modeler (FPMOD) as previously described (37). Molecular models for CFP-SLN and YFP-SLN were constructed similarly using the NMR structure determined for human SLN (PDB accession 1JDM), fused to the x-ray crystal structures of CFP (PDB accession 1OXD) and YFP (PDB accession 1YFP).…”

“…Baculovirus Expression-CFP-SLN and YFP-SLN were expressed as previously described for CFP-SERCA1a and YFP-PLB (37,42,43). Spodoptera frugiperda (Sf21) insect cells were cultured in shaker flasks at 27°C in the dark.…”

Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells

“…1A shows a schematic representation of the F-L577 protein constructed in this study to investigate the conformational changes linked with SERCA2a activity in living cells. Because the N-terminal fusion of ECFP to SERCA1a has no notable effects on its ATPase activity (35), ECFP was fused to the N terminus of the A-domain of SERCA2a (Fig. 1A).…”