A LEUCINE-ZIPPER STABILIZES THE PENTAMERIC MEMBRANE DOMAIN OF PHOSPHOLAMBAN AND FORMS A COILED-COIL PORE STRUCTURE

Phospholamban is a phosphoprotein regulator of cardiac sarcoplasmic re ticulum which is phosphorylated in response to beta-adrenergic stimula tion. Previous results have shown that phospholamban forms Ca2+-select ive channels in lipid bilayers. The channel-forming domain has been lo calized to amino acid residues 26-52, which form a stable pentameric, helical structure. The specific residues responsible for stabilizing t he pentameric membrane domain of phospholamban have been identified by mutational analysis. Residues 26-52 were individually mutated to Ala or Phe, and the ability of the resulting mutant to form a pentamer or other oligomer was assessed by SDS-polyacrylamide gel electrophoresis analysis. Replacement of Leu(37), Ile(40), Leu(44), Ile(47), or Leu(51 ) by Ala prevented pentamer formation, indicating their essential invo lvement in the oligomeric assembly. The heptad repeats, and 3-4-residu e spacing of the essential amino acids suggest that residues 37-52 ado pt a pentameric coiled-coil structure stabilized by a leucine zipper m otif formed by the close packing of Leu(37), Ile(40), Leu(44), Ile(47) , and Leu(51). The resulting symmetric structure contains a central po re defined by the hydrophobic surface of the five stabilizing leucine zippers, which are oriented to the interior and form the backbone of t he pentamer.