Abstract
Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.
Highlights
These changes in structure and dynamics of PLN induced by single mutations have direct effects on its inhibitory potency
In the absence of SERCA, PLN is in equilibrium between a more populated T state where the cytoplasmic domain is in direct contact with the lipid membrane and a less populated R state
Unphosphorylated PLN binds SERCA to form an inhibitory complex, whereas Ser-16 phosphorylation leads to a non-inhibitory complex without completely dissociating from the enzyme
Summary
NMR Sample Preparation—15N-Labeled monomeric PLN mutants were expressed as a fusion protein with maltose-binding protein and purified as described previously (38). NMR Titrations of PLN Mutants with SERCA: Determination of Kd and T to R Transitions—NMR titrations were carried out monitoring the chemical shifts of the amide fingerprint regions of PLN variants using [1H,15N] HSQC experiments. The Kd values were determined by following the disappearance of the resonances corresponding to domain II of PLN upon addition of incremental amounts of SERCA as described previously (13, 35). Unlike conventional spin labels attached flexibly to cysteine side chains, this spin label couples the nitroxide moiety rigidly to the ␣ carbon, providing direct correlation to peptide backbone dynamics This spin label has no effect on PLN function (48). Vmax was obtained from the fit, and the data were plotted as V/Vmax versus pCa. Because the maximal velocity is not reproducible for the SERCA1⁄7PLN complex, we used the pKCa values to quantify the inhibition caused by PLN mutants
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