Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.
ABSTRACT Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca(2+) affinity of SERCA2a by competing for Ca(2+) binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca(2+)-ATPase activity and E1 approximately P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca(2+) concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca(2+) for binding to SERCA2a. This was confirmed with the Ca(2+) pump mutant, D351A, which is catalytically inactive but retains strong Ca(2+) binding. Increasingly higher Ca(2+) concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca(2+) binding. Finally, the specific conformation of E2 (Ca(2+)-free state of SERCA2a) that binds PLB was investigated using the Ca(2+)-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca(2+) showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca(2+) binding to SERCA2a by stabilizing a unique E2.ATP state that is unable to bind thapsigargin or vanadate.
SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Phospholamban (PLB) is a pentameric protein that plays an important role in regulating cardiac contractility via a reversible inhibitory association with the sarcoplasmic reticulum Ca2+ATPase (SERCA), the enzyme responsible for maintaining correct calcium homeostasis. Here we study the functional and biophysical characteristics of a PLB mutant associated with human dilated cardiomyopathy (DCM), with a deletion of arginine at position 14 (PLBR14Δ). In agreement with recent findings, we find that PLBR14Δ has a reduced inhibitory effect on SERCA compared to wild type PLB (PLBWT) when reconstituted into lipid membranes. The mutation also leads to a large reduction in the protein kinase A-catalysed phosphorylation of Ser-16 in the cytoplasmic domain of PLBR14Δ. Measurements on SERCA co-reconstituted with an equimolar mixture of PLBWT and PLBR14Δ (representing the lethal heterozygous state associated with DCM) indicates that the loss-of-function mutation has a dominant effect on PLBWT functionality and phosphorylation capacity, suggesting that mixed PLBWT/PLBR14Δ pentamers are formed that have characteristics typical of the mutant protein. Structural and biophysical analysis of PLBR14Δ indicates that the mutation perturbs slightly the helical structure of the PLB cytoplasmic domain and reduces its affinity for the phospholipid bilayer surface, thereby altering the orientation of the cytoplasmic domain relative to the wild-type protein. These results indicate that the structure and function consequences of the R14 deletion have profound effects on the regulation of SERCA which may contribute to the aetiology of DCM.PLoS ONE 09/2014; 9(9):e106746. DOI:10.1371/journal.pone.0106746 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Phospholamban (PLN) is a key regulator of cardiac contraction and relaxation through its inhibition of the sarco/endoplasmic reticulum Ca2 +-ATPase (SERCA2a). The inhibitory effect is attenuated upon protein kinase A (PKA) dependent phosphorylation of PLN. PLN exists in an equilibrium of pentamers and monomers. While monomers inhibit SERCA2a by direct interaction the function of the pentamers is still unclear. Here we tested the hypothesis that the PLN pentamer exhibits an important regulatory role by modifying PKA-dependent phosphorylation of inhibitory monomeric PLN subunits. Using Western blot analyses and antibodies specific for PKA-dependent phosphorylation of PLN, pentamers showed stronger signals than monomers both in transfected HEK293 cells and cardiomyocytes. Upon activation of PKA, phosphorylation of protomers in the PLN pentamers increased faster and at lower levels of stimulation than PLN monomers suggesting pentamers as the preferred PKA target. The comparison of phosphorylation patterns at different pentamer / monomer ratios revealed that pentamers delay phosphorylation of PLN monomers. A mechanistic explanation was provided by co-immunoprecipitation that suggested high affinity of PKA for PLN pentamers. Both monomers and pentamers were pulled down with SERCA2a indicating co-localization. Unlike pentamers, phosphorylated PLN monomers fully dissociated from the Ca2 +-ATPase upon stimulation of PKA. These findings suggest a model where PLN pentamers reduce phosphorylation of monomers at baseline and delay monomer phosphorylation upon PKA stimulation leading to increased interaction of PLN monomers with SERCA2a.Journal of Molecular and Cellular Cardiology 01/2015; 80. DOI:10.1016/j.yjmcc.2014.12.020 · 5.22 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca(2+)-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca(2+)-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K(+) and 3 mM Mg(2+). Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K(+) ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K(+) in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H(+)771. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca(2+)-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.Biophysical Journal 04/2015; 108(7):1697-1708. DOI:10.1016/j.bpj.2015.03.004 · 3.83 Impact Factor