title: Competitive displacement of wild-type phospholamban from the Ca2+-free cardiac calcium pump by phospholamban mutants with different binding affinities.
abstract: Phospholamban (PLB) regulates the cardiac Ca(2+) pump (SERCA2a). To test how different species of PLB mutants compete to interact with the Ca(2+)-free, E2 conformation of SERCA2a, using the insect cell expression system, we examined how various exogenous PLB mutants regulated SER-20G-PLB, a chimeric WT-SERCA2a-Gly-WT-PLB construct, which retains a fully catalytic active Ca(2+)-pump and its intrinsically regulatory PLB-tether. Exogenous gain-of-function PLB mutants dominantly super-inhibited the WT-PLB-tethered SERCA2a. Further, in the Ca(2+)-free condition, co-expressed normal- or super-inhibitory PLB mutant with either engineered N30C or V49C residue cross-linked to Lys328 or V89C of SER-20G-PLB at the cytoplasmic or transmembrane domain, respectively, suggesting that these freely diffusing PLB mutants completely replaced the WT-PLB-tether and fit into the binding pocket previously occupied by WT-PLB. Micromolar Ca(2+) completely inhibited cross-linking, yielding a similar Ca(2+)-dependency regardless of the presence of the WT-PLB-tether. In contrast, the PLB mutant with the loss-of-function L31A mutation has decreased binding affinity for SERCA2a, thus cross-linking weakly to the WT-PLB-tethered SERCA2a, and only marginally affected the activity of SER-20G-PLB. Thus, there is a reversible equilibrium between different PLB mutants for binding to E2, in which PLB mutants possessing higher binding affinity for SERCA2a produce a more stable E2·PLB and lower Ca(2+) affinity.
authors.affiliation: Krannert Institute of Cardiology and the Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: zhechen@iu.edu.
authors.forename: Zhenhui
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Binding affinity; Cardiac Ca(2+)-ATPase; Cross-linking; Enzyme regulation; Equilibrium; Phospholamban