Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2 x ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2 x TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca2+ (E1 x Ca2) or adopts the E2P or E2 x TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.