Reexamination of the role of the leucine/isoleucine zipper residues of phospholamban in inhibition of the Ca2+ pump of cardiac sarcoplasmic reticulum.

Phospholamban is a small phosphoprotein inhibitor of the Ca(2+)-pump in cardiac sarcoplasmic reticulum, which shows a distinct oligomeric distribution between monomers and homopentamers that are stabilized through Leu/Ile zipper interactions. A two-faced model of phospholamban inhibition of the Ca(2+)-pump was proposed, in which the Leu/Ile zipper residues located on one face of the transmembrane alpha-helix regulate the pentamer to monomer equilibrium, whereas residues on the other face of the helix bind to and inhibit the pump. Here we tested this two-faced model of phospholamban action by analyzing the functional effects of a new series of Leu/Ile zipper mutants. Pentameric stabilities of the mutants were quantified at different SDS concentrations. We show that several phospholamban mutants with hydrophobic amino acid substitutions at the Leu/Ile zipper region retain the ability to form pentamers but at the same time give the same or even stronger (i.e. L37I-PLB) inhibition of the Ca(2+)-pump than do mutants that are more completely monomeric. Steric constraints prevent the Leu/Ile zipper residues sequestered in the interior of the phospholamban pentamer from binding to the Ca(2+)-pump, leading to the conclusion that the zipper residues access the pump from the phospholamban monomer, which is the active inhibitory species. A modified model of phospholamban transmembrane domain action is proposed, in which the membrane span of the phospholamban monomer maintains contacts with the Ca(2+)-pump around most of its circumference, including residues located in the Leu/Ile zipper region.

Phosphorylation-induced structural change in phospholamban and its mutants, detected by intrinsic fluorescence.

We have used intrinsic fluorescence to test the hypothesis that phosphorylation induces a conformational change in phospholamban (PLB), a regulatory protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB, which relieves inhibition of the cardiac Ca-ATPase, has been shown to decrease the mobility of PLB in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the present study, we found that this mobility shift depends on the acrylamide concentration in the gel, suggesting that phosphorylation increases the effective Stokes radius. To further characterize this structural change, we performed spectroscopic experiments under the conditions of SDS-PAGE. CD indicated that phosphorylation at Ser-16 does not change PLB's secondary structure significantly. However, the fluorescence of Tyr-6 in the cytoplasmic domain of PLB changed significantly upon PLB phosphorylation: phosphorylation increased the fluorescence quantum yield and decreased the quenching efficiency by acrylamide, suggesting a local structural change that decreases the solvent accessibility of Tyr-6. A point mutation (L37A) in the transmembrane domain, which disrupts PLB pentamers and produces monomers in SDS-PAGE and in lipid bilayers, showed similar phosphorylation effects on fluorescence, indicating that subunit interactions within PLB are not crucial for the observed conformational change in SDS. When PLB was reconstituted into dioleoylphosphatidylcholine (DOPC) lipid bilayers, similar phosphorylation effects in fluorescence were observed, suggesting that PLB behaves similarly in response to phosphorylation in both detergent and lipid environments. We conclude that phosphorylation induces a structural change within the PLB protomer that decreases the solvent accessibility of Tyr-6. The similarity of this structural change in monomers and pentamers is consistent with models in which the PLB monomer is sufficient for the phosphorylation-dependent regulation of the Ca-ATPase.

Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers.

Phospholamban (PLB), a 52-residue protein integral to the cardiac sarcoplasmic reticulum, is a key regulator of the Ca pump. PLB has been shown to form pentamers in the denaturing detergent sodium dodecyl sulfate (SDS), but its oligomeric state in the natural environment of the lipid membrane remains unknown. In order to address this issue, we performed electron paramagnetic resonance (EPR) experiments on two types of lipid-reconstituted, recombinant PLB: wild type (WT PLB) and a mutant substituted with alanine at leucine 37 (L37A PLB), whose propensity to oligomerize in SDS is greatly diminished. The lipid used in reconstitution was dioleoylphosphatidylcholine (DOPC) doped with a phospholipid 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 oligomeric (effective oligomeric size of 3.52 +/- 0.71), while L37A PLB is monomeric (effective oligomeric size of 1.15 +/- 0.15). Thus, the oligomeric states of these proteins in the lipid membrane are remarkably similar 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 the cardiac Ca pump, changes the lipid-PLB interactions, decreasing the number of lipids restricted by contact with protein. The results are consistent with a phosphorylation-dependent increase of the effective oligomer 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 phosphorylation due to reduced electrostatic repulsion among PLB protomers.

Effects of membrane thickness on the molecular dynamics and enzymatic activity of reconstituted Ca-ATPase.

We have studied the effect of phospholipid chain length on the activity and molecular dynamics of reconstituted Ca-ATPase from skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence anisotropy (TPA) and electron paramagnetic resonance (EPR). We used reconstituted Ca-ATPase in exogenous phosphatidylcholines with monounsaturated chains 14-24 carbons long, to determine their effects on the physical properties of the Ca-ATPase and to correlate these physical changes with changes in the ATPase activity. In agreement with previous studies, we found that the enzyme activity was maximal with a chain length of 18 and decreased substantially with longer or shorter chains. Our TPA results show that chain lengths longer or shorter than the optimal 18 result in a significantly decreased mobility of the Ca-ATPase, indicated by higher residual anisotropy and suggesting extensive protein aggregation. Saturation-transfer EPR data obtained with a spin label bound to a different site also indicates substantial immobilization of the enzyme, supporting the TPA results. There is good agreement between the fractional inhibition of the Ca-ATPase activity and the fraction of the enzyme in large aggregates. Solubilization in the nonionic detergent C12E8 demonstrated that inhibition of enzyme activity is reversible. In contrast to the large effects on protein mobility, these changes in chain length had little or no effect on hydrocarbon chain mobility as detected by conventional EPR at different depths in the membrane. We conclude that the Ca-ATPase has an optimum lipid bilayer thickness, presumably matching the thickness of the hydrophobic transmembrane surface of the enzyme, and that deviation from this optimum thickness produces a hydrophobic mismatch that induces protein aggregation and hence Ca-ATPase inhibition. This is consistent with our proposal that protein dynamics and protein-protein interactions are of primary importance to the Ca-ATPase mechanism.