Helical structure of phospholamban in membrane bilayers

Abstract

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca2+ pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-13C]Leu7 and [3-13C]Ala11 in the cytoplasmic region, between [1-13C]Pro21 and [3-13C]Ala24 in the juxtamembrane region and between [1-13C]Leu42 and [3-13C]Cys46 in the transmembrane domain of phospholamban are consistent with α-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long α-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca2+ pump.