Mutation and Phosphorylation Change the Oligomeric Structure of Phospholamban in Lipid Bilayers

Abstract

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.

Functional Co-expression of the Canine Cardiac Ca2+Pump and Phospholamban in Spodoptera frugiperda (Sf21) Cells Reveals New Insights on ATPase Regulation

“…To examine this issue, SERCA2a was co-expressed with L37A-PLB, which is monomeric on SDS-PAGE (17) and, more importantly, also when reconstituted in lipid membranes (19). Surprisingly, we observed that L37A-PLB was a more effective suppressor of SERCA2a Ca 2ϩ affinity than wild-type phospholamban.…”

“…Microsomes isolated from infected Sf21 cells exhibit high levels of ATP hydrolysis and active Ca 2ϩ transport, and, furthermore, cardiac-like coupling between phospholamban and SERCA2a is retained. With the baculovirus system, we also demonstrate that a monomerforming mutant of phospholamban (17,19), unexpectedly, is a stronger inhibitor of SERCA2a activity than is the pentamer, suggesting that the monomer may be the key molecular species regulating the Ca 2ϩ pump in sarcoplasmic reticulum membranes. No evidence for a Ca 2ϩ channel activity of phospholamban was obtained.…”

“…We detected no differences in coupling coefficients between microsomes expressing SERCA2a alone, or microsomes expressing SERCA2a plus phospholamban. (17) or when reconstituted in phospholipid membranes (19). Since L37A-PLB is essentially depolymerized in lipid membranes, we co-expressed it with SERCA2a in Sf21 cells to test if the phospholamban monomer is sufficient for inhibition of the ATPase.…”

“…We recently reported on the use of this system for the expression and mass purification of canine cardiac phospholamban and several of its protein mutants from Sf21 1 cells (16,17). The purified, recombinant protein was successfully reconstituted into proteoliposomes to study its secondary structure (18) and oligomeric organization in the lipid bilayer (19). Successful co-reconstitution with Ca 2ϩ pumps purified from rabbit skeletal muscle (16) and canine myocardium (20) was also achieved.…”

Quantification of protein–lipid selectivity using FRET

“…M13 MCP is composed of a single transmembrane segment and as such is expected to immobilize around 10–12 phospholipids in its immediate vicinity or annular binding sites (Marsh and Horváth 1998; Cornea et al. 1997). However, due to MCP aggregation, only five annular binding sites were identified by protein molecule when using ESR (Wolfs et al.…”