Both acidic and basic amino acids in an amphitropic enzyme, CTP:phosphocholine cytidylyltransferase, dictate its selectivity for anionic membranes.
Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.
Johnson
Joanne E
JE
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Xie
Mingtang
M
Singh
Laila M R
LM
Edge
Robert
R
Cornell
Rosemary B
RB
D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024361:Amino Acids, Basic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D051381:Rats
D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D007527:Isoenzymes; D008563:Membrane Lipids; D010455:Peptides; D011522:Protons; D019916:Choline-Phosphate Cytidylyltransferase