CONFORMATION AND LIPID-BINDING PROPERTIES OF 4 PEPTIDES DERIVED FROM THE MEMBRANE-BINDING DOMAIN OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE

We are probing the mechanism of the lipid selective membrane interacti ons of CTP: phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a conti nuous amphipathic ct-helix between residues similar to 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examin ed the secondary structure and membrane binding properties of syntheti c peptides derived from domain M: a 62mer peptide encompassing the ent ire domain (Pep62), a 33mer corresponding to the N-terminal portion (P epNH1), and two 33mers corresponding to the three C-terminal 11mer rep eats, one with the wild-type sequence (Pep33Ser), and one with the thr ee serines in the nonpolar face substituted with alanine (Pep33A1a). P eptide secondary structure was analyzed by circular dichroism, and lip id interactions were analyzed by a direct vesicle binding assay, by ef fects of lipid vesicles on peptide tryptophan fluorescence, and by mon olayer surface pressure changes. All peptides bound to vesicles as alp ha-helices with selectivity for anionic lipids. Binding involved inter calation 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 bin d 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 similar to 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupt ing the nonpolar face of the amphipathic helix serve to lower the lipi d affinity and thereby enhance selectivity for anionic lipids. Althoug h diacylglycerol is an activator of the enzyme, none of the peptides r esponded differentially to PC/diacylglycerol vesicles versus pure PC v esicles, suggesting that domain M alone is not sufficient for the enzy me's response to diacylglycerol. Increases in surface pressure at an a ir-water interface indicated that the domain M peptides had strong sur face-seeking tendencies. This supports a binding orientation for domai n M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting conde nsation 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).