NMR Solution Structure and Topological Orientation of Monomeric Phospholamban in Dodecylphosphocholine Micelles

Abstract

Phospholamban is an integral membrane protein that regulates the contractility of cardiac muscle by maintaining cardiomyocyte calcium homeostasis. Abnormalities in association of protein kinase A with PLB have recently been linked to human heart failure, where a single mutation is responsible for dilated cardiomyopathy. To date, a high-resolution structure of phospholamban in a lipid environment has been elusive. Here, we describe the first structure of recombinant, monomeric, biologically active phospholamban in lipid-mimicking dodecylphosphocholine micelles as determined by multidimensional NMR experiments. The overall structure of phospholamban is "L-shaped" with the hydrophobic domain approximately perpendicular to the cytoplasmic portion. This is 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.

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A31P,2H, and13C Solid-State NMR Spectroscopic Study

“…The two peaks are well within the range of an α-helical structure (see Table 1). The observed α-helical structure of the cytoplasmic domain of PLB agrees with the L-shaped (see Figure 1(A)) and the bellflower-like assembly high-resolution solution NMR structures (see Figure 1(C)) and disagrees with the non-α-helical disordered cytosolic domain model shown in Figure 1(B) (57,60,61).…”

“…Finally, it is important to emphasize that the high-resolution NMR solution structures of the monomeric PLB mutant (AFA-PLB) and the pentameric WT-PLB in micelles reported by the Veglia and Chou groups, respectively, represent two of the best and widely accepted membrane protein structures available in the literature (60,61). Additionally, EPR spectroscopic studies from the Veglia and Thomas groups indicate that a two-state dynamic equilibrium exists for the cytosolic segment of AFA-PLB in a lipid bilayer (58,65,66).…”

Spectroscopic validation of the pentameric structure of phospholamban

“…and D.D.T. groups using solution NMR (1,(11)(12)(13)(14) and EPR in detergent micelles (13), EPR in lipid vesicles (13,15,34), and solid-state NMR in mechanically oriented lipid bilayers (18). In all of these experiments, the results are unambiguous: the major population of AFA-PLN is L-shaped.…”

“…conformation of the monomer is L-shaped, with the cytoplasmic domain in contact with the surface of the lipid bilayer (1,(15)(16)(17)(18).…”

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

“…Recent developments in bacterial expression systems for the preparation of recombinant isotopically labeled membrane proteins, methods for sample preparation, pulse sequences for high-resolution spectroscopy, and structural indices that guide the structure assembly process, have greatly extended the capabilities of these techniques, and the structures of a variety of helical membrane proteins have been determined by NMR in micelles and in bilayers [3][4][5][6][7][8][9][10][11].…”