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.
Bibliographical noteFunding Information:
This work was supported in part by grants to G.V. (National Institutes of Health GM64742; American Heart Association 0160465Z), and D.D.T. (National Institutes of Health GM27906; University of Minnesota Academic Health Center).
The authors thank R. Di Fonzo for helpful comments; D. Live and B. Ostrowski for assisting with the NMR experiments; T. Krick and Dr. L.A. Higgins for assistance in the Facility for Mass Spectrometry in the Life Sciences at the University of Minnesota; and C. Karim and T. Kirby for helpful discussions. We benefited from the outstanding facilities at the Minnesota High-Field NMR Center in the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota. NMR instrumentation was provided with funds from the National Science Foundation (BIR-961477) and the University of Minnesota Medical School. The coordinates for all phospholamban conformers have been deposited in the Protein Data Bank database ( http://www.rcsb.org/pdb/index.html ); PDB ID code: 1N7L.