Conformation and Lipid Binding Properties of Four Peptides Derived from the Membrane-Binding Domain of CTP:Phosphocholine Cytidylyltransferase (original) (raw)

We are probing the mechanism of the lipid selective membrane interactions of CTP: phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic R-helix between residues ∼240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as R-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only ∼1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule). CTP:phosphocholine cytidylyltransferase (CT), 1 a key ratedetermining enzyme for PC biosynthesis, is regulated by reversible membrane binding. Membrane binding and enzyme activation can be regulated by fluctuations in the membrane content of acidic lipid, and/or diacylglycerol (DAG), or by changes in the phosphorylation state of CT (1-3). Activation of the enzyme in vitro by lipid vesicles is dependent on the mole percent of acidic lipid or DAG mixed with PC (4-8). Diacylglycerol or enzyme dephosphorylation lowers the percent of acidic lipid required for activation (3, 9). Both electrostatic and hydrophobic interactions mediate the membrane binding process (7-10). Mammalian CT is organized into at least three discrete domains. The N-terminal two-thirds of the protein forms a protease-insensitive domain (11), which has been proposed to house the catalytic site on the basis of homologies to other cytidylyltransferases (12-14), and mutational analysis (15, 16). The C-terminal region of the protein is highly phos