We have characterized the structural determinants of homo-oligomerization for phospholamban (PLB) and sarcolipin (SLN), using experimental and computational assays. PLB and SLN are transmembrane peptides that regulate contractility via phosphorylation-dependent regulation of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA). 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). We tested self-assembly of the tetrameric C41L mutants of PLB, demonstrating that the C41L-PLB tetramer (1) is stabilized by residues on a- and d- positions of the heptad-repeat, similar to pentameric WT-PLB, (2) reverts to a WT-like pentamer when the I48A mutation is added to the same e -position as C41L, and (3) is not stabilized by a potential zipper on the native b -position of the heptad repeat. Thus, PLB e -position residues in the cleft between subunits contribute to self-association affinity and stoichiometry, in addition to a - and d -position residues. We also tested self-assembly of SLN, identifying two residues (V14, L21) and a novel a -position heptad repeat that contribute to self-association affinity and stoichiometry, in addition to the d -position that was previously identified for SLN oligomerization. Molecular dynamics simulations were performed to test for channel formation and stability of homo-oligomers of SLN and PLB. We propose that PLB and SLN populate a distribution of oligomeric forms in SR membranes (monomer through pentamer and hexamer). The results suggest that pore formation, which has been observed in vitro, requires increased toroidal stoichiometry. The University of Minnesota Supercomputing Institute provided computational resources. Funding was provided by NIH R01HL139065 (to D.D.T./R.T.R.).
Svensson et al. (2026) studied this question.