Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions

Abstract

Phospholamban (PLN) is a single-pass membrane protein that regulates the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Phosphorylation of PLN at Ser16 reverses its inhibitory function under β-adrenergic stimulation, augmenting Ca2+ uptake in the sarcoplasmic reticulum and muscle contractility. PLN exists in two conformations; a T state, where the cytoplasmic domain is helical and absorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies from our group have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR techniques to compare the structural topology and conformational dynamics of monomeric PLN (PLNAFA) with that of the PLNR14del, a naturally occurring deletion mutant that is linked to the progression of dilated cardiomyopathy. We found that the behavior of the inhibitory transmembrane domain of PLNR14del is similar to that of the native sequence. In contrast, the conformational dynamics of R14del both in micelles and lipid membranes are enhanced. We conclude that the deletion of Arg14 in the cytoplasmic region weakens the interactions with the membrane and shifts the conformational equilibrium of PLN toward the disordered R state. This conformational transition is correlated with the loss-of-function character of this mutant and is corroborated by SERCA’s activity assays. These findings further support our hypothesis that SERCA function is fine-tuned by PLN conformational dynamics and begin to explain the aberrant regulation of SERCA by the R14del mutant.

Probing membrane protein ground and conformationally excited states using dipolar- and J-coupling mediated MAS solid state NMR experiments

“…Recent progress on solid-state NMR of membrane proteins have demonstrated the importance of using both dipolar- and J-coupling mediated pulse sequences for complete structural mapping 2,28,30,39,57. Phospholamban is a typical example for the application of these methods to demonstrate the change in conformational equilibrium using 1D and 2D experiments 26,27,53,58 . Similar pulse sequences using both 13 C- and 15 N-detected CP- and rINEPT-based experiments have also been applied to various single- and multi-pass membrane proteins including sarcolipin, α-synuclein, bacterial chemotaxis receptor, GPCR, and BamA complex 16,28,59–63 .…”

Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

“…The proposed mechanisms for superinhibition were an imbalance between the pentameric and monomeric PLN forms, resulting in an increase of the monomeric (inhibitory) one [7]. Further studies did not confirm a superinhibitory effect of PLN p.Arg14del in the basal state, but suggested that it might result from an impaired response of mutant PLN to PKA-mediated phosphorylation [8,9]. Although Ca 2+ cycling abnormalities have been detected in cardiac myocytes carrying the mutation [10,11], it remains altogether unclear whether they indeed reflect SERCA2a superinhibition.…”

Istaroxime treatment ameliorates calcium dysregulation in a zebrafish model of phospholamban R14del cardiomyopathy

“…The PLN R14del mutation results in a protein that has a stronger affinity for SERCA2a 6 , which has been correlated with the disruption of the Ser 16 phosphorylation motif. It was suggested that disruption of this motif could lead to an increased inhibition of the SERCA2a, cytoplasmic Ca 2+ overload, and increased risk for malignant ventricular arrhythmias 17 , 18 . Whether changes in intracellular Ca 2+ dynamics also occur in vivo and affect cardiac structure and function remains unaddressed.…”