Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase

Chemical synthesis, functional reconstitution, and electron paramagnetic re sonance (EPR) have been used to analyze the structure and function of phosp holamban (PLB), a 52-residue integral membrane protein that regulates the c alcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-P AGE, EPR, and fluorescence. It has been proposed that inhibition of the pum p is due primarily to the monomeric form, with both pentameric stability an d inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-P AGE, and compared it with the synthetic null-cysteine TM domain (residues 2 6-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isol ated TM domain is more oligomeric than the full-length parent molecule. The se results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic Thf domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic doma ins (residues 1-25). When reconstituted into Liposomes containing the Ca-AT Pase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule, We conclude that the TM domain of PLB i s sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.