Phospholamban does not Interact with E2 Intermediates of the Cardiac Calcium Pump Stabilized by Metal Fluorides

Abstract

The phospholamban (PLB) monomer binds in the groove between M2, M4, and M9 of the cardiac calcium pump (SERCA2a) and inhibits enzyme activity. We previously demonstrated that cross-linking of PLB with engineered Cys-residues to this region is prevented by micromolar Ca, but is stimulated by the nucleotides ATP or ADP. We proposed that PLB inhibits the catalytic activity of the enzyme by stabilizing a unique, nucleotide bound, Ca-free intermediate of SERCA2a (E2•ATP). Stabilization of E2•ATP prevents Ca binding at the high affinity Ca binding sites and formation of E1, thus blocking the kinetic cycle. To further test our model, we have now checked if PLB interacts with other E2 conformations, notably, SERCA2a with the bound metal fluorides (MgF42-, AlF4-, or BeF3-), which mimic the E2•Pi, E2P, and E2-P states, respectively. Interactions between PLB and SERCA2a were examined by cross-linking of N30C-PLB to K328 of WT-SERCA2a at the cytoplasmic extension of M4 and of V49C-PLB to V89C-SERCA2a at the C-terminal end of M2. Insect cell microsomes co-expressing N30C-PLB/SERCA2a or V49C-PLB/V89C-SERCA2a were first incubated with the metal fluorides in Ca-free buffer, and then subjected to chemical cross-linking. All these metal fluorides prevented cross-linking of PLB to SERCA2a at both the cytoplasmic and transmembrane regions, suggesting that PLB does not interact with E2•Pi, E2P, and E2-P formed in the absence of nucleotide. Remarkably, however, the cross-linking of PLB to SERCA2a was completely restored upon addition of 3mM ATP, allowing formation of E2•ATP. These results strongly support our model in which E2•ATP is the single conformation binding PLB, and further suggest that the binding interaction between E2•ATP and PLB is very strong, sufficient to prevent formation of E2•Pi, E2P, or E2-P, even when stabilized by metal fluorides.