The PLM Homotetramer has a Structural Basis that Parallels that of PLB: The Leucine Zipper

Abstract

Phospholemman (PLM or FXYD1) interacts with and inhibits the sodium potassium ATPase (mainly by reducing its Na affinity), an effect that is relieved by PLM phosphorylation. This is analogous to phospholamban (PLB) regulation of SERCA. Like PLB, PLM is thought to also form homo-oligomers, although the structural basis for this oligomerization is still unknown. Here we use both a computational and FRET approach to address this. Alanine substitutions of leucine and isoleucine residues in the PLM transmembrane segment were examined for their effect on PLM-PLM FRET. We found that substitutions at I23, I26, L30 and L33 all significantly reduce FRET, but not so for I29 and L36. In parallel experiments, we used Rosetta to model the PLM oligomer. Mapping of experimental data onto Rosetta models favored tetramer configuration for this oligomer rather than a trimer or pentamer. In the tetramer model, the I23, I26, L30 and L33 residues all face and interact with an adjacent PLM subunit, whereas the I29 and L36 residues face the center of the tetramer and appear unlikely to be involved in the stabilization of the tetramer structure. Additionally, the core of the tetramer is lined with hydrophobic residues and is spatially constricted, suggesting that the PLM tetramer does not function as a channel. We conclude that the PLM homo-oligomer is a tetramer with a structural basis that parallels that of PLB: the leucine zipper.