Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

Krannert Institute of Cardiology and the Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2010; 285(37):28540-52. DOI: 10.1074/jbc.M110.151779
Source: PubMed


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.

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    • "We have used a variety of techniques to investigate the functional and conformational properties of PLBWT and the mutant PLBR14Δ lacking R14 in the cytoplasmic domain. It is generally accepted that PLBWT reduces the Km of SERCA for calcium, but it has been variably shown to decrease or increase Vmax or to have no effect [32], [35], [41]–[44]. Here we find that PLBR14Δ is a slight loss of function mutant that is capable of interacting with SERCA and lowering its affinity for calcium, but to a lesser degree than PLBWT alone (Figure 2). "
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    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.
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    ABSTRACT: To study PLB (phospholamban) inhibition of the cardiac Ca(2+) pump [SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a)], a fusion protein (SER-20G-PLB) was engineered by tethering SERCA2a with PLB through a 20-glycine residue chain, allowing the PLB tether to either bind to or dissociate from the inhibition site on SERCA2a. When expressed in insect cells, SER-20G-PLB produced active Ca(2+) uptake, which was stimulated by the anti-PLB antibody, both similar to that which occurred with the control sample co-expressing WT (wild-type)-SERCA2a and WT-PLB. The K(Ca) values of Ca(2+)-dependent ATPase were similar for SER-20G-PLB (0.29±0.02 μM) and for the control sample (0.30±0.02 μM), both greater than 0.17±0.01 μM for WT-SERCA2a expressed alone. Thus SER-20G-PLB retains a fully active Ca(2+) pump, but its apparent Ca(2+) affinity was decreased intrinsically by tethered PLB at a 1:1 molar stoichiometry. Like WT-PLB, SER-20G-PLB ran as both monomers and homo-pentamers on SDS/PAGE. As Ca(2+) concentrations increase from 0 to the micromolar range, the proportion of non-inhibiting pentamers increased from 32% to 52%, suggesting that Ca(2+) activation of the pump completely dissociates the PLB tether from the inhibition site on SERCA2a, with concurrent association of PLB pentamers. Collectively, the regulation of SERCA2a is achieved through the Ca(2+)-dependent equilibria involving PLB association and dissociation from SERCA2a, and assembling and disassembling of SER-20G-PLB pentamers.
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    ABSTRACT: To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.
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