Spectroscopic function-structure analysis of the SERCA-PLB complex

Abstract

Cardiomyocyte contraction is controlled by intracellular Ca<super>2+</super> concentrations. Action potential opens the voltage-gated calcium channel in the sarcolemma and triggers the calcium-induced calcium-release mechanism to release Ca<super>2+</super> stored in the sarcoplasmic reticulum (SR) through ryanodine receptors. For muscle relaxation to occur, Ca<super>2+</super> must be removed from the cytosol. Most of the activator Ca<super>2+</super> is sequestered back into the SR by sarco(endo)plasmic reticulum Ca<super>2+</super>-ATPase (SERCA). In ventricular myocytes, SERCA is regulated by a small integral membrane protein, phospholamban (PLB). PLB binds and inhibits SERCA, and this inhibition is physiologically relieved by either micromolar Ca<super>2+</super> in systole, or by phosphorylation at Ser16 or Thr17 through &beta;-adrenergic stimulation. A decline in SERCA activity is implicated in heart failure irrespective of etiologies. Recent gene therapies for heart failure emphasize enhancing SERCA activity by decreasing PLB inhibition. However, the structural mechanism of relief of inhibition still remains elusive. This thesis work is motivated to elucidate the structural basis for SERCA regulation by PLB, hence providing more information for the design of next generation gene and drug therapies.This thesis work uses time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the structures of the SERCA-PLB complex in its activated or inhibited forms. In the first project, we investigated the function effect of the equilibrium of PLB cytoplasmic domain between an ordered R state and a disordered T state on SERCA regulation. We varied the lipid headgroup charges to perturb this equilibrium through electrostatic interactions with the positively charged PLB cytoplasmic domain. TR-FRET measurements, in conjunction with functional data and electron paramagnetic resonance experiments, established the correlation of the T/R equilibrium with PLB inhibitory potency. In the second project, we studied the structures of the SERCA-PLB complex under physiological conditions that relieve inhibition. TR-FRET distance measurements between cytoplasmic domains of SERCA and PLB revealed that phosphorylation of PLB at Ser16 relieves SERCA inhibition mainly by shifting the T/R equilibrium toward the less inhibitory R state, and partially by dissociating the complex. Micromolar Ca<super>2+</super> probably relieves inhibition through structural rearrangements within the transmembrane domain of the complex. In the last project discussed in this thesis, we used western blot to quantify different phosphorylation states of PLB in pig cardiac SR. PLB can be phosphorylated at either Ser16 or Thr17, generating four phosphorylation states: unphosphorylated, phosphorylated only at Ser16, phosphorylated only at Thr17, or phosphorylated at both sites. We also found that each PLB phosphorylation state has a distinct inhibitory potency for SERCA.