Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.
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.
Arkin
I T
IT
Department of Cell Biology, Yale University School of Medicine, New Haven, CT.
Adams
P D
PD
MacKenzie
K R
KR
Lemmon
M A
MA
Brünger
A T
AT
Engelman
D M
DM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D006801:Humans; D007473:Ion Channels / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000099211:Phospholamban
D000596:Amino Acids; D001704:Biopolymers; D002135:Calcium-Binding Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000099211:Phospholamban