STRUCTURAL ORGANIZATION OF THE PENTAMERIC TRANSMEMBRANE ALPHA-HELICESOF PHOSPHOLAMBAN, A CARDIAC ION-CHANNEL

Phospholamban is a 52 amino acid calcium regulatory protein found as p entamers in cardiac SR membranes. The pentamers form through interacti ons between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed inter actions between the transmembrane segments, using a chimeric protein c onstruct in which staphylococcal nuclease (a monomeric soluble protein ) is fused to the N-terminus of phospholamban. The chimera forms penta mers observable in SDS-PAGE, allowing the effects of mutations upon th e oligomeric association to be determined by electrophoresis. The disr uptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Re sidues of the same class lined up on faces of a 3.5 amino acids/turn h elical projection, allowing the construction of a model of the interac ting surfaces in which the helices are associated in a left-handed pen tameric coiled-coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily for m a left-handed coiled-coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show cons iderable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channe ls. The cylindrical ion pore (minimal diameter of 2 Angstrom) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.