Toward a high-resolution structure of phospholamban: design of soluble transmembrane domain mutants.

Determination of a high-resolution structure of the phospholamban (PLB) transmembrane domain by X-ray crystallography or NMR is handicapped by the hydrophobic nature of the peptide. Interestingly, the crystal structure of the five-stranded parallel coiled-coil oligomerization domain from cartilage oligomeric matrix protein (COMPcc) shows marked similarities to a model proposed for the pentameric transmembrane domain of PLB. Contrary to the putative coiled-coil domain of PLB, COMPcc contains mostly hydrophilic amino acids on the surface, resulting in a soluble molecule. Here, we report the design of soluble PLB transmembrane domain variants by combining the surface residues of COMPcc and the hydrophobic interior of the transmembrane domain of PLB. The soluble PLB variants formed pentameric structures as revealed by analytical ultracentrifugation. After redox shuffling, they showed unspecific disulfide bridge patterns similar to that of the chemically synthesized wild-type PLB transmembrane domain. These results suggest a structural homology between the soluble PLB mutants and the wild-type PLB transmembrane domain. Together with the data reported in the literature, they furthermore indicate that residues Leu37, Ile40, Leu44, and Ile47 of the PLB sequence specify pentamer formation. In contrast, a designed recombinant COMPcc mutant, COMP-ARCC, which was engineered to contain the two PLB cysteines that potentially could form an interchain disulfide bridge, formed a specific disulfide bond pattern. This finding indicates structural differences between the transmembrane domain of PLB and COMPcc. The soluble PLB variants may be used to determine a high-resolution structure of the PLB pentamer by X-ray crystallography.