MUTATION AND PHOSPHORYLATION CHANGE THE OLIGOMERIC STRUCTURE OF PHOSPHOLAMBAN IN LIPID BILAYERS

Phospholamban (PLB), a 52-residue protein integral to the cardiac sarc oplasmic reticulum, is a key regulator of the Ca pump. PLB has been sh own to form pentamers in the denaturing detergent sodium dodecyl sulfa te (SDS), but its oligomeric state in the natural environment of the l ipid membrane remains unknown. In order to address this issue, we perf ormed electron paramagnetic resonance (EPR) experiments on two types o f lipid-reconstituted, recombinant PLB: wild type (WT PLB) and a mutan t substituted with alanine at leucine 37 (L37A PLB), whose propensity to oligomerize in SDS is greatly diminished. The lipid used in reconst itution was dioleoylphosphatidylcholine (DOPC) doped with a phospholip id spin-label that detects protein contact. EPR spectroscopy was used to determine the fraction of the total lipid molecules in contact with PLB. Our results show that, in phospholipid bilayers, WT PLB is oligo meric (effective oligomeric size of 3.52 +/- 0.71), while L37A PLB is monomeric (effective oligomeric size of 1.15 +/- 0.15). Thus, the olig omeric states of these proteins in the lipid membrane are remarkably s imilar to those in SDS solution. In particular, the point mutation in L37A PLB greatly destabilizes the PLB oligomer. Phosphorylation of PLB by protein kinase A, which has been shown to relieve inhibition of th e cardiac Ca pump, changes the lipid-PLB interactions, decreasing the number of Lipids restricted by contact with protein. The results are c onsistent with a phosphorylation-dependent increase of the effective o ligomer size of WT PLB from 3.52 to 5.34 and of L37A PLB from 1.15 to 1.91. These phosphorylation effects were abolished in a medium with a high ionic strength. We conclude that the oligomeric states of PLB in Lipid membranes are in a dynamic equilibrium that is perturbed by phos phorylation due to reduced electrostatic repulsion among PLB protomers .