Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2

Abstract

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.

Cα‐Methyl proline: A unique example of split personality

“…Similar results [b-turn formation and trans L-Xxx-L-(aMe)Pro peptide bonds] were reported for L-(aMe)Pro-containing analogues of an antigen mimotope peptide 29 and the antimicrobial peptide buforin 2. 30 Interestingly, in contrast to the results from molecular mechanics simulations, it was experimentally found that the sequence -L-(aMe)Pro-L-Pro is not tightly folded. 31 Finally, a recent X-ray diffraction analysis of two terminally blocked dipeptides of general formula Ac-L-Val-L-Xxx-NHR [where Xxx is 4-methylene-(aMe)Pro] clearly indicates the absence of any intramolecular H-bond.…”

Cationic Antimicrobial Peptides

“…The good antimicrobial activity (Table 15), but inability to permeabilize the bacterial membranes, is consistant with a membrane-independent mechanism of action as previous studies have suggested [46,167,169]. which has a structural similarity to MEL [166].…”

“…BUF is not membrane active and the peptide appears to target intracellular nucleic acids after translocation across lipid bilayers without significant permeabilizing activity [167]. A hinge induced by a P residue within the sequence was found to be responsible for translocation across the cell membranes [46].…”

Membrane-translocating peptides and toxins: from nature to bedside

“…For instance, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flipflop. 35 Interestingly, the hybrid buforin II-magainin 2, in which the proline hinge region of buforin II was fused to the amino-terminal helix of magainin 2, is able to translocate the bacterial membrane and delivery into the cytoplasm the antimicrobial -helical portion of magainin 2.Also, it was demonstrated that the proline-rich antimicrobial peptide Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy-and temperaturedependent process, probably by macropinocytosis and direct membrane translocation. Additional, investigations also reveal the intracellular uptake of Bac7(1-35) by 3T3 cells is enhanced during S phase, suggesting a novel function for this proline-rich peptide.…”

“…For instance, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flipflop. 35 Interestingly, the hybrid buforin II-magainin 2, in which the proline hinge region of buforin II was fused to the amino-terminal helix of magainin 2, is able to translocate the bacterial membrane and delivery into the cytoplasm the antimicrobial -helical portion of magainin 2.…”