CHAPTER 22: Structural Topologies of Phosphorylated and Non-phosphorylated Oligomeric Phospholamban in Lipid Membranes by a Hybrid NMR Approach
Published:24 Feb 2014
V. Vostrikov and G. Veglia, in Advances in Biological Solid-State NMR: Proteins and Membrane-Active Peptides, ed. F. Separovic and A. Naito, The Royal Society of Chemistry, 2014, pp. 425-443.
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Phospholamban (PLN) is a single-pass membrane protein that regulates Ca2+ transport into the cardiac sarcoplasmic reticulum (SR) by binding the SR Ca2+-ATPase (SERCA). Non-phosphorylated PLN decreases SERCA's apparent Ca2+ affinity, reducing uptake in the SR. Phosphorylation reverses the inhibition, augmenting the relaxation of the cardiac muscle. In lipid membranes and micelles, both phosphorylated and non-phosphorylated PLN assemble into pentamers, whose biological role is under debate. Using a hybrid NMR approach, including solution, oriented, and magic angle spinning techniques, we determined the structures and membrane topology of both phosphorylated and non-phosphorylated PLN pentamers. The pentamers adopt a pinwheel topology, with the transmembrane helices assembled in a left-handed coiled-coil quaternary structure. These transmembrane domains form a narrow pore of ∼2 Å widths and ∼25 Å lengths held together by a Leu/Ile zipper. The dimension of this central pore does not enable the passage of ions, such as Ca2+ or Cl−, and remains invariant upon phosphorylation. Thus, oligomerization regulates SERCA by tuning the concentration of PLN available to SERCA, thereby keeping the ATPase within a physiological window of inhibition. This integrative NMR approach is applicable to other membrane proteins, where multiple structural biology and biophysical approaches are necessary to elucidate their biological function.