The N-Terminal Region of a Ph-Responsive Peptide Controls Its Interaction with Phosphatidylserine-Containing Bilayers

Abstract

Local acidosis is common in solid tumors, and provides a promising target for peptide-based anticancer treatments. To exploit this characteristic, the acidity-triggered rational membrane (ATRAM) peptide was designed to insert selectively into lipid bilayers when the surrounding media is acidic. Insertion of ATRAM into the bilayer as a transmembrane α-helix involves translocation of the C-terminus across the membrane, such that the N-terminus is free to interact with lipids on the outer leaflet. Since many cancers overexpose on the outer membrane leaflet the negatively charged lipid phosphatidylserine (PS), we propose that mutations at the non-inserting N-terminus would modify ATRAM's interactions with model cancer cell membranes. Here, we used two ATRAM variants, termed K2-ATRAM (L2K/L5K) and Y-ATRAM (L2Y), to explore the effects of positively charged or mildly hydrophobic amino acids near the N-terminus. The presence of PS in the membrane enhanced membrane partitioning for K2-ATRAM but decreased it for Y-ATRAM. However, screening with high-salt conditions showed that the effects of PS on the pK of insertion are multifactorial and cannot explained by electrostatics alone. These results suggest that studies using only neutral bilayers may fail to indicate the true interactions of anticancer peptides with tumor cells, which contain negatively charged phospholipids. Additionally, the different behaviors of the ATRAM variants demonstrate how the non-inserting N-terminal region can be tailored to adjust the specificity of ATRAM for target membranes. This information will aid the design of new ATRAMs and other peptides that are tailored to the pH and lipid compositions of a variety of diseases.