Structural Dynamics and Topology of Phosphorylated Phospholamban Homopentamer Reveal Its Role in the Regulation of Calcium Transport

Abstract

Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and non-phosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation of each monomer. The TM domains form a hydrophobic pore of approximately 24 Å long, and 2 Å in diameter, which is inconsistent with canonical Ca2+ selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing the partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window.

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

“…The large span of the 15 N anisotropic chemical shifts and 15 N- 1 H dipolar couplings indicate that the helical axis is tilted by approximately 30-35° with respect to the normal of the lipid bilayer. Previous studies from our group showed that the tilt angle of PLN WT is dependent on the lipid bilayer thickness as well as the choice of the alignment system – mechanical or magnetic [13]. Similar behavior is observed for the monomeric species, where the tilt angle in DMPC:POPC bicelles is some 10-15° lower than the one in DOPC:DOPE glass slides.…”

“…The majority of the changes are localized near the deletion site and propagate from the juxtamembrane domain Ib to the transmembrane domain II, with Ser16 and Glu19 exhibiting the largest perturbations. These effects resemble the case of S16 phosphorylation, where the local chemical shift perturbations are associated with the unfolding of the domain Ia helix and concomitant detachment of this domain from the membrane, leading to an increase of the local flexibility [13]. To confirm that the structural changes manifested by the shift in the resonance frequencies correspond to changes in the conformational dynamics, we measured T 1 , T 2 and [ 1 H, 15 N]-NOE for the backbone amide groups.…”

“…In past years we have used NMR spectroscopy to determine PLN structures free and bound to SERCA [13, 19, 20]. Based on our studies, we proposed a regulatory model in which PLN conformational equilibrium is central to SERCA regulation.…”

Selenoprotein K form an intermolecular diselenide bond with unusually high redox potential

“…It is also quite possible that, like other bitopic proteins, it is capable of forming both homo- and hetero-oligomers. Many bitopic proteins dynamically exchange between different oligomerization states [50]. The close proximity of Sec from opposing monomers will facilitate the formation of diselenide bonds across the dimer interface.…”

Atomistic insights into human Cys-loop receptors by solution NMR

“…With the use of solution NMR, several studies revealed K + and pH-dependent conformational and dynamics changes of KcsA [56-60]. The structural dynamics and topology of the homopentameric phospholamban (PLN) channel (~31 kDa) were determined using a combination of solution and solid-state NMR methods to reveal the role of PLN in the regulation of calcium transport [61, 62]. More recently, an unusual architecture of the 42-kDa homo-hexameric p7 channel, the hepatitis C virus viroporin, was revealed using the latest solution NMR technologies [63].…”