Abstract 12676: C-Terminal Residues of Phospholamban are Critical Determinants of the Quaternary Structure and Function of the Calcium ATPase Regulatory Complex

Abstract

Rationale: A naturally occurring missense Leu-39stop (L39X) mutation in phospholamban (PLB) results in truncation of the C-terminal transmembrane domain, leading to cardiomyopathy and premature death in humans. Objective: The goal of this study was to determine the structural and regulatory role of the C-terminal residues of PLB in the membranes of living cells. Methods and Results: We fused fluorescent protein tags to PLB and cardiac Ca 2+ ATPase (SERCA) to investigate the role of PLB C-terminal residues for membrane localization, PLB oligomerization and SERCA regulation. Alanine substitution of C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB. Notably, substitution mutation V49A had profound effects on pentamer structure and regulatory complex conformation, increasing and decreasing probe separation distance, respectively. Progressive deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. Selective permeabilization of the plasma membrane by saponin resulted in diffusion of fluorescently labeled PLB out of the cells, consistent with solubilization of truncated proteins. Molecular dynamics simulations recapitulated decreased bilayer anchoring for truncated PLB. C-terminal truncations resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the SERCA-PLB binding affinity, and loss of inhibitory potency as quantified by Ca 2+ -dependent ATPase activity. However, despite decreased SERCA-PLB binding, intermolecular FRET was paradoxically increased as a result of a 14.5 Å decrease in the distance between donor and acceptor fluorophores. Conclusions: We conclude that PLB C-terminal residues are critical for membrane anchoring and quaternary structure determination of PLB pentamer and PLB-SERCA regulatory complex. The loss of membrane registration restraint by C-terminal residues (especially V49) causes displacement of PLB to an alternative position on SERCA. The data are compatible with a model in which PLB binds to the canonical inhibitory binding site and an additional novel site.