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.
Bibliographical noteFunding Information:
This research was supported by NIH ( R01GM64742 , R01GM072701 , 1S10OD021536 to G.V., R01GM27906 and R37AG26160 to D.D.T.) and F31HL095361 to K.N.H. S.E.D.N. was supported by NIHT32AR07612 (to D.D.T.), and subsequently by the American Heart Association ( 16PRE27770056 ). NMR experiments were carried out at the Minnesota NMR Center.
- Ca2+ regulation
- Conformational equilibrium
- Dilated cardiomyopathy
- Solid-state NMR