Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation |
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Authors: | Xiaoqiong Dong David D Thomas |
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Institution: | 1. Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA;2. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA |
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Abstract: | We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca2+ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and Ca2+]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca2+ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca2+ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex. |
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Keywords: | IAF 5-iodoacetamidofluorescein DOPC 1 2-dioleoyl-sn-glycero-3-phosphocholine DOPE 1 2-dioleoyl-sn-glycero-3-phosphoethanolamine EGTA ethylene glycol-bis-(2-aminoethylether)-N N N&prime N&prime -tetraacetic acid ESI-MS electrospray ionization mass spectroscopy FRET fluorescence resonance energy transfer TR-FRET time-resolved FRET MOPS 3-(N-morpholino)propanesulfonic acid pCa &minus log10[Ca2+] pKCa &minus log(KCa) calcium concentration at half-maximal ATPase activity PLB phospholamban SERCA sarco(endo)plasmic reticulum Ca2+-ATPase SR sarcoplasmic reticulum OG octyl β-d-glucopyranoside PKA protein kinase A |
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