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

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 adsorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR to compare the structural topology and conformational dynamics of monomeric PLN (PLN(AFA)) with that of the PLN(R14del), 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 PLN(R14del) is similar to that of the native sequence. Conversely, 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 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.