Mutations Linked to Dilated Cardiomyopathy Perturb the Topology and Regulatory Response of Phospholamban

Abstract

Phospholamban (PLN), a small 52-residue membrane protein expressed in cardiac tissue, functions as a regulatory subunit to the 110 kDa sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) calcium pump. The SERCA-PLN complex is responsible for restoring high SR Ca2+ concentrations prior to each cardiomyocyte contraction, in which PLN maintains SERCA's activity within a tight physiological window. PLN binds SERCA via a transmembrane protein-protein interaction that suppresses the affinity of SERCA's Ca2+ sites, but this effect is relieved upon phosphorylation of PLN's cytoplasmic extension by protein kinase A (PKA) upon β-adrenergic stimulation. PLN mutations that alter its activity or interfere with phosphorylation are linked to hereditary forms of dilated cardiomyopathy (DCM), although the reasons for this remain unclear due to a lack of structural data for the SERCA-PLN complex. Using structural restraints from high-resolution magic-angle spinning (MAS) and oriented sample (OS) solid-state NMR (ssNMR) spectroscopy, we have constructed dynamic models of the SERCA-PLN complex that capture a topological allosteric mechanism for PLN-driven communication of cytoplasmic regulatory stimuli to SERCA's Ca2+ binding sites. Further OS-ssNMR measurements and activity assays of DCM mutants of PLN displayed an altered topology and a dampened response to phosphorylation, which was consistent with our dynamic models and suggest disease onset may occur from an impaired β-adrenergic pathway. Overall, our recent observations by OS-ssNMR and modelling demonstrate that structural topology (i.e., helical tilt, rotation and secondary structure) is a critical determinant of SERCA regulation by PLN as well as by other homologous regulatory partners expressed in non-cardiac tissues.