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

Abstract

Approximately, 70% of the Ca ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca-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 Ca 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.

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells

“…Some of the PLN mutations have opposite sub-cellular mechanisms of action. One of them, R9C, results in chronic inhibition of SERCA2a by mutant PLB and early death, which is consistent with the known mechanisms on PLB regulation and the findings in mice 48 , 51 . The other mutation is associated with loss of PLB function (L39stop) and results in dilated cardiomyopathy and premature death in the homozygous, opposite to what is seen in mice 50 .…”

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

“…Other PLN variants support these findings, showing that the population of these high energy states can be shifted by phosphorylation, genetic or engineered mutations (R14 deletion, and S16E,T17E), or lipid composition. Although it is difficult to determine the absolute R-state population, we utilized the relative integrated intensities from the rINEPT and CP experiments to obtain a semi-quantitative estimate of the R-state population 27,53 . Figure 2 shows the comparison of the 1D 15 N signal intensities for CP and rINEPT spectra, which display significant changes in the conformational equilibrium among different PLN variants.…”

“…When reconstituted in zwitterionic DMPC lipids, the R9C mutant shows the lowest R-state population (0.3%), whereas the R14del and R25C mutants display 20 and 25% R state population, respectively. The R state population of PLN, which is about 3% in DMPC, can be enhanced up to 6 and 25% by incorporating the positively charged lipids, ePOPC, which interacts with the positively charged Lys and Arg residues of the cytoplasmic region of PLN 27,53 .…”

The phenotypic characteristic observed by cardiac magnetic resonance in a PLN-R14del family

“…(3) Failing to be phosphorylated and regulated by PKA, or further, disabling the normal function of PKA 6,10,11 . For instance, R9C is an Arg-Cys mutation which occurs in the cytoplasmic region (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). This region is critical for PKA to recognize and bind PLN.…”

“…The systole of cardiomyocytes is triggered by the rhythm of calcium sparks released by RyR2, and the diastole is delicately regulated by SERCA, which uptakes intracellular calcium back to sarcoplasmic reticulum 2 . Dynamic control of SERCA activity relies on the phosphorylation and dephosphorylation, as well as several conformational changes of phospholamban (PLN), a 52-acid protein 3 , 4 . PLN attaches SERCA to form a complex to inhibit the ability of SERCA, and the inhibition is mainly released by the phosphorylation by PKA.…”