NMR solution structure of phospholamban

Phospholamban (PLN),an amphipathic intrinsic membrane protein of 52 amino a cids, is the modulator of the Ca2+ pump of cardiac, slow-twitch, and smooth -muscle sarcoplasmic reticulum. In response to beta-adrenergic stimulation, it becomes phosphorylated at Ser(16) and/or Thr(17) and dissociates from t he pump, which, in turn, achieves its full activity. Here we present the th ree-dimensional structure of chemically synthesized, monomeric PLN in an or ganic solvent. Monomerization (PLN normally forms homopentamers) was obtain ed by replacing Cys(41) With phenylalanine (Phe = Fl, a modification that d id not affect biological activity. The structure was determined by high-res olution NMR in CHCl3/MeOH of the unphosphorylated state of [F-41]PLN (C41F) . Of the hydrophilic cytoplasmic parts IA (Met(1) to Pro(21)) and IB (Gln(2 2) to As-30) and the membrane-spanning hydrophobic domain II (Leu(31) to Le u(52)) of PLN, domain IA, which contains the two phosphorylation sites Ser( 16) and Thr(17), and domain II have been suggested to be helical and connec ted through the less-structured hinge-region IB. In the structural study pr esented here, [F-41]PLN is composed of two alpha-helical regions connected by a beta-turn (type III). The residues of the beta-turn (type III) are Thr (17), Ile(18), Glu(19), and Met(20), the first being one of the two phospho rylation sites (Ser(16) and Thr(17)). The hinge region is located at the C- terminal end of domain IA, and domain IB is part of a second helix. The two alpha-helices comprising amino acids 4-16 and 21-49 are well-defined (the root-mean-square deviations for the backbone atoms, calculated for a family of the structures, are 0.58 and 0.92 Angstrom, resp.). Pro(21) is at the b eginning of the C-terminal helix and in the trans conformation.