Phospholamban and Sarcolipin Share Similar Transmembrane Zipper Motifs That Control Self-Association Affinity and Homo-Oligomer Stoichiometry

We characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-assembly, using experimental and computational assays. PLB and SLN are transmembrane peptides that regulate contractility via phosphorylation-dependent regulation of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). The physiological role of PLB and SLN has been proposed to be important as a reservoir for fine-tuning SERCA activity and to act as ion-selective channels. Structural studies of PLB and SLN transmembrane domains have indicated stabilization of higher-order oligomers by leucine/isoleucine zippers in a heptad-repeat motif (a–g residue positions), requiring residue C41. Here we have modeled and tested the additional residues responsible for self-assembly using alanine replacement. We demonstrate that PLB e-position residues in the cleft between subunits contribute to self-association affinity and stoichiometry. For SLN, we identified two residues (V14, L21) and a novel heptad repeat (a-position) that contribute to self-association affinity and stoichiometry. This is in addition to the d-position that we previously identified for SLN oligomerization. Our molecular models demonstrate stable hexamer assemblies, but without the likelihood of a hydrated pore. We propose that PLB and SLN populate a distribution of oligomeric forms in sarcoplasmic reticulum membranes (monomer through pentamer and hexamer) and suggest that pore formation requires increased toroidal stoichiometry.