Phospholamban (PLN), a 52-residue integral membrane protein, is the endogenous regulator of Ca-ATPase in cardiac muscle. When in its unphosphorylated form, PLN inhibits Ca-ATPase and translocation of calcium into the sarcoplasmic reticulum (SR). Inhibition of the enzyme activity is relieved upon phosphorylation at Ser16 or at micromolar calcium concentrations in the cytosol.

PLN consists of cytoplasmic and transmembrane domain helices connected by a semi-flexible loop. The cytoplasmic domain helix is in contact with the surface of the lipid bilayer, forming an overall L-shape or bent structure. The wild-type PLN (WT-PLN) is believed to exist as a homo-pentamer (260-residues), but interacts and inhibits Ca-ATPase as a monomer. For this reason, our previous studies have focused on a fully functional monomeric mutant of PLN (AFA-PLN), however, recently we have begun studies using WT-PLN.

Summary

Solid-state NMR spectroscopy, in conjunction with rigid body molecular dynamics calculations, shows that monomeric phospholamban in lipid bilayers has two distinct helical domains, with an interhelical angle within 60-100 degrees, ruling out the possibility of a continuous alpha-helical structure for this protein.

Summary

The protocol described in this paper outlines the technique used for expressing and purifying recombinant PLN and sarcolipin (SLN) from Escherichia coli bacteria. Fusions of PLN and SLN to maltose binding protein (MBP) were constructed for protein expression and subsequent purification, facilitating large-scale production of highly pure protein. The regulation of Ca-ATPase activity by recombinant PLN and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLN and SLN.

Summary

The overall structure of PLN was solved in DPC detergent micelles, revealing the structure to be L-shaped with the hydrophobic domain approximately perpendicular to the cytoplasmic portion, in agreement with our previously published solid-state NMR data. In addition, there are two striking discrepancies between our structure and those reported previously for synthetic phospholamban in organic solvents: a) in our structure, the orientation of the cytoplasmic helix is consistent with the amphipathic nature of these residues; and b) within the hydrophobic helix, residues are positioned on two discrete faces of the helix as consistent with their functional roles ascribed by mutagenesis. This topology renders the two phosphorylation sites, Ser-16 and Thr-17, more accessible to kinases.

Summary

PLN is composed of three structural domains: a transmembrane domain from residues 22 to 52, a connecting loop from 17 to 21, and a cytoplasmic domain from 1 to 16 that is organized in an L-shaped structure where the transmembrane and the cytoplasmic domain form an angle of approximately 80 degrees. This publication, using T1, T2, and 1H-15N nuclear Overhauser effect values, measured amide backbone dynamics, revealing the existence of four dynamic domains, showing residues 22-30 within PLN to be more dynamic than the rest of the transmembrane domain. We propose that these dynamic properties are critical factors in the biomolecular recognition of PLN by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.

Summary

Dipolar waves are distinct hallmarks of both the secondary and tertiary structures of alpha-helical proteins that are immobilized in membrane bilayers or embedded in anisotropic media. We present a simple, semi-empirical approach that exploits the modulation of the amplitude and average of dipolar waves to determine the topology of alpha-helical proteins. Moreover, we describe the application of this method for the structural determination of a detergent solubilized membrane protein, PLN that is involved in calcium regulation of cardiac muscle. When combined with high-resolution solid-state NMR data, this method can serve as a fast route for determining the topology of helical membrane proteins solubilized in detergent micelles.

Summary

Our data show that phosphorylation at Ser16 of PLN disrupts the L-shaped structure of monomeric PLN, causing significant unwinding of both the cytoplasmic helix (domain Ia) and the short loop (residues 17-21) connecting this domain to the transmembrane helix (domains Ib and II). Concomitant with this conformational transition, we also find pronounced changes in both fast and slow time scale dynamics upon phosphorylation. We propose that the regulatory mechanism of PLN phosphorylation involves an order-to-disorder transition, resulting in a decrease in the PLN inhibition of Ca-ATPase.

Summary

We have used magnetic resonance to map the interaction surface of PLN to Ca-ATPase. To map the molecular details of the PLN/Ca-ATPase interaction, we have functionally reconstituted SERCA with labeled PLN in DPC detergent micelles for high-resolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. Based on structural and dynamics data, we propose a model in which PLN undergoes allosteric activation upon encountering SERCA.

Summary

PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Here, we show that the single phosphoryl transfer at Ser16 induces a more pronounced conformational switch to the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA is not affected (K(d) values for the transmembrane domains of pPLN and PLN are approximately 60 microM), supporting the hypothesis that phosphorylation at Ser16 does not dissociate PLN from SERCA. However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, we propose that these sites remotely control the conformation of domain Ib. These findings constitute a paradigm for how post-translational modifications such as phosphorylation in the cytoplasmic portion of membrane proteins control intramembrane protein-protein interactions.