Characterizing Phospholamban to Sarco(endo)plasmic Reticulum Ca2+-ATPase 2a (SERCA2a) Protein Binding Interactions in Human Cardiac Sarcoplasmic Reticulum Vesicles Using Chemical Cross-linking

Abstract

Background:The mechanism of PLB inhibition of SERCA2a activity remains unresolved. Results: Cross-linking Lys 27 of PLB to SERCA2a in human SR vesicles is conformationally dependent and correlates closely with enzyme inhibition. Conclusion: PLB stabilizes the E2⅐ATP state, thereby blocking formation of E1. Significance: Mutually exclusive binding of PLB and Ca 2ϩ regulates SERCA2a activity in normal and failed human myocardium.

The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

“…This alteration in the apparent Ca 2ϩ affinity of SERCA is the hallmark of PLB inhibition (2, 3). As expected, addition of the 2D12 anti-PLB antibody (which mimics the effect of Ser 16 / Thr 17 phosphorylation of PLB (23)) largely reversed SERCA inhibition by WT-PLB and PLB4 (Fig. 3B, shaded …”

“…The supernatant was collected and PLB was added from the concentrated working solutions at a ratio of 0.14 mg of PLB/1.0 mg of solubilized SR vesicle protein, determined in control experiments to be a saturating concentration of PLB for inhibition of Ca 2ϩ -ATPase activity by lowering the apparent Ca 2ϩ affinity. This amount of added PLB gave a molar ratio of PLB to SERCA of 2.9:1, as determined by quantitative immunoblotting (16). Final volumes of mother liquors were adjusted by addition of 20% glycerol to make the final EGTA concentration 2 mM and samples were stored at 4°C.…”

“…PLB inhibits SERCA activity by decreasing Ca 2ϩ affinity at its two Ca 2ϩ binding sites (I and II) located in the SR membrane, thereby attenuating SR Ca 2ϩ filling and contractile force development. Phosphorylation of PLB at Ser 16 and Thr 17 partially reverses PLB induced alterations in Ca 2ϩ affinity, substantially augmenting contractility (2-4). Chemical cross-linking was used previously to localize the PLB binding site on SERCA to a groove formed between transmembrane helices M2, M4, M6, and M9 of the Ca 2ϩ pump (5)(6)(7)(8)(9).…”

Hydrophobic Imbalance in the Cytoplasmic Domain of Phospholamban Is a Determinant for Lethal Dilated Cardiomyopathy

“…Considering the suggestion that PLN dissociates from SERCA following calcium binding (48,49), the simulations support a physical interaction that persists at least through all three calcium binding steps of the reaction cycle (Fig. 2).…”

Synthetic Phosphopeptides Enable Quantitation of the Content and Function of the Four Phosphorylation States of Phospholamban in Cardiac Muscle

“…increases K Ca , the free Ca 2ϩ concentration corresponding to half-maximal SERCA activation) and contributes to contractile dysfunction (5,6). Phosphorylation at Ser-16 and/or Thr-17 partially reverses this inhibition by decreasing K Ca (5,(7)(8)(9)(10). Spectroscopic studies show that PLB phosphorylation induces structural rearrangement within the PLB-SERCA complex without dissociating PLB, which is essentially a subunit of SERCA under physiological conditions (11)(12)(13)(14).…”