Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

• Published in: Biochimica et biophysica acta. Biomembranes 2018-06, Vol.1860 (6), p.1335-1341
• Main Authors: Nelson, Sarah E.D., Ha, Kim N., Gopinath, Tata, Exline, Mara H., Mascioni, Alessandro, Thomas, David D., Veglia, Gianluigi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Ca2+ regulation; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Cardiomyopathy, Dilated - genetics; Conformational equilibrium; Consensus Sequence; Dilated cardiomyopathy; Humans; Lipid Bilayers - chemistry; Membrane Proteins - chemistry; Membrane Proteins - genetics; Mutation, Missense; Nuclear Magnetic Resonance, Biomolecular; Phospholamban; Point Mutation; Protein Conformation; Protein Domains; Protein Structure, Secondary; Recombinant Proteins - metabolism; Sarcoplasmic Reticulum Calcium-Transporting ATPases - metabolism; SERCA; Solid-state NMR
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2018.02.030

Approximately, 70% of the Ca2+ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca2+ binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA. [Display omitted] •Solid-state NMR reveals the conformational equilibrium of phospholamban in lipid membranes.•Arg9Cys mutation drives the equilibrium toward the T state of phospholamban.•Arg25Cys mutation shifts the conformational equilibrium toward the R state.•Changes in membrane interactions might be linked to the dysfunctional regulation of SERCA by phospholamban.