Different Content of Charged Residues at the N-termini of Pore-forming Sticholysin I (StI) and Sticholysin II (StII) are Responsible for Different Interactions with Air-Water and Lipid-Water Interfaces
Carlos Alvarez1,2; Diana Martínez1; Fábio Turra3; Uris Ros1; Edson Crusca3; Aisel Valle1; Amaury Pupo1; Eduardo M. Cilli3; María E. Lanio1; and Shirley Schreier2
1Center for Protein Studies, University of Havana, Cuba; 2Institute of Chemistry, USP, São Paulo, and 3Institute of Chemistry, UNESP, Araraquara, Brazil
The sea anemone isotoxins StI and StII have 93% sequence identity. The only three non-conservative amino acid substitutions occur in the N-terminal region, involving charged residues. The most noticeable difference between both toxins is StII's higher hemolytic activity. However, intrinsic Trp fluorescence data and permeabilization assays show higher StI activity in model membranes. To investigate possible causes of this difference, four peptides from the N-terminus of StI (SELAGTIIDGASLTFEVLDKVLGELGKVSRK, P1-31 and P12-31) and StII (ALAGTIIAGASLTFQVLDKVLEELGKVSRK, P1-30 and P11-30) were synthesized and their interaction with interfaces was examined using a microtensiometer. The surface activity (SA) of StI at the air-water interface was higher than that of StII. Correspondingly, P1-31 elicited higher surface tension than P1-30. In contrast, no relevant differences were found between P12-31 and P11-30, which exhibited SA similar to that of P1-31. In addition, StI had higher critical pressure (pc) than StII in phosphatidylcholine (PC), sphingomyelin (SM) and PC:SM (1:1) monolayers. The association of the basic toxins decreased upon inclusion of negatively charged phosphatidic acid (PA) into PC:SM monolayers. The decrease was larger for StI than for StII, suggesting that the N-terminus is important for toxin-monolayer interaction. The introduction of PA did not change P1-31 pc, but caused a reduction of P1-30 pc. For all lipid systems, while the latter peptide behaved similarly to StII in terms of pc and slopes of the binding isotherms, P1-31 showed lower pc and a greater slope than StI, suggesting a different accommodation of the peptide in the monolayer. The similar behavior of the peptides lacking the first ten residues emphasizes the role of E2 and D9 in the differential interaction of P1-31 and P1-30 with monolayers and suggests that differences in the N-terminus play an important role in toxin-membrane interaction.
Supported by FAPESP, CNPq, USP.
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