Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.

Abstract

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N‐terminus of phospholamban. The chimera forms pentamers observable in SDS‐PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left‐handed pentameric coiled‐coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form 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 considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) 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.

Structure of the Transmembrane Cysteine Residues in Phospholamban

“…Unlike the acetylcholine receptor, in which the polar residues appear to line the ion channel, the cysteine residues in phospholamban are distributed around the transmembrane helix. Mutagenesis and S-H exchange measurements indicate that C41 is oriented towards the surrounding lipids and C46 is packed in the helix interface (Arkin et al, 1994;Arkin et al, 1996). C36 is the only cysteine that contributes to the accessible surface of the channel, although S-H exchange measurements indicate that the sulfhydryl is protected.…”

“…All calculations were carried out on the model of phospholamban arrived at by molecular dynamics and mutagenesis studies previously described (Arkin et al, 1994;Adams et al, 1995). Hydrogen atoms were introduced using the CHARMM22 parameter set (Brooks et al, 1983) with electrostatic and van der Waals energy terms enabled using the program X-PLOR (Brünger, 1992), in order to insure energetically reasonable initial positions for the hydrogen atoms.…”

“…The electrostatic field is likely to be a contributing element in establishing the specificity and conductivity of the channel. This idea was explored by calculating the electrostatic potentials in the channel using the structural model of phospholamban (Arkin et al, 1994;Arkin et al, 1996). It is important to state that the electrostatic calculations were undertaken for a model of phospholamban and not a structure determined by crystallographic or NMR methods.…”

“…Structurally, phospholamban has been modeled as a left-handed coiled-coil of five long helies (Adams et al, 1995;Arkin et al, 1994Arkin et al, , 1996. The cytosolic region of the helical bundle contains the two residues (S16 and T17) that are phosphorylated in response to ␤-adrenergic stimulation (Tada & Kadoma, 1989).…”

“…The cytosolic region of the helical bundle contains the two residues (S16 and T17) that are phosphorylated in response to ␤-adrenergic stimulation (Tada & Kadoma, 1989). Specific interactions between transmembrane helices hold the complex together and form a pore through the bilayer (Arkin et al, 1994;Adams et al, 1995). Interspersed between the hydrophobic residues in the transmembrane domain of phospholamban are three cysteine residues (C36, C41 and C46).…”