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1.C.16 The Magainin (Magainin) Family

Many organisms synthesize proteins (or peptides) which are degraded to relatively small hydrophobic or amphipathic, bioactive peptides. These peptides exhibit antibiotic, fungicidal, virucidal, hemolytic and/or tumoricidal activities by interacting with membranes and forming transmembrane channels that allow the free flow of electrolytes, metabolites and water across the phospholipid bilayers. Most of these peptides appear to function in biological warfare. There are many designations given to these bioactive peptides. They include the magainins, cecropins, melittins, defensins, bacteriocidins, etc. The proteins in each family within this functional superfamily are homologous, but they exhibit little or no significant sequence similarity with members of the other families. Thus, each family may have evolved independently. However, certain common structural features observed between members of distinct families suggest that at least some of these families share a common ancestry. Pore size and subunit stoichiometry of the pore have been determined (Watanabe and Kawano 2016).  Cardiolipin prevents membrane-pore formation by magainin and the human cathelicidin LL-37 in phosphatidyl glycerol membranes, and this constitutes a plausible mechanism used by bacteria such as Staphylococcus aureus to act against stress perturbations and, thereby, gain resistance to antimicrobial agents (Rocha-Roa et al. 2021).

Magainins are a group of amphipathic peptides (21-26 amino acyl residues in length) from the skin and intestines of frogs. They form right-handed α-helical structures in membranes and serve bactericidal, fungicidal and virucidal functions. They thereby provide defense against infectious agents. As revealed by their synergistic behavior, they can form homo- or heterooligomeric transmembrane channels. One distant member of the magainin family, 'peptide between glycine and leucine amide' (PGLa) (GMASKAGAIAGKIAKVALKAL-NH2) is not hemolytic but acts on Gram-negative and Gram-positive bacteria as well as fungi and protozoa. PGLa is positively charged, adopts a random coil configuration in water and folds into an amphipathic α-helix when it inserts into the membrane, parallel to the membrane surface at low peptide concentrations but perpendicular to the membrane surface at higher concentrations when it forms a peptide/lipid pore. Pore formation depends on anionic phospholipids. PGLa and magainin2 amide exhibit synergism when forming transmembrane pores. The synergism of magainin 2 amide and PGLa in forming transmembrane pores results from formation of a heterodimer composed of parallel helices with strong membrane permeabilizing activity (Hara et al., 2001; Nishida et al., 2007). While magainin 1 is anion-selective, magainin 2 is cation-selective (Kourie and Shorthouse, 2000).

The antimicrobial peptides magainin 2 and the PGLa (PYLa) precursor (TC# 1.C.16.1.5) show marked synergism. These two peptides form a heterodimer composed of parallel helices with strong membrane permeabilizing activity (Hara et al., 2001). The synergy observed for the two peptides is due to the formation of a parallel heterodimer (Nishida et al., 2007). Magainin disruption of cells is concentration-dependent and a highly stochastic process (Han et al., 2009). Based on NMR and other analyses, Han et al., 2009 concluded that magainin-induced pores in lipid vesicles have a mean diameter of approximately 80 A.

Model membranes have been used to elucidate how peptides permeabilize membranes. The interaction of F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS), an equipotent analogue of magainin 2 isolated from the African clawed frog Xenopus laevis was studied with unfixed Bacillus megaterium and Chinese hamster ovary (CHO)-K1 cells by Imura et al. (2008). The influx of fluorescent markers of various sizes into the cytosol revealed that magainin 2 permeabilizes bacterial and mammalian membranes in significantly different ways. The peptide formed pores with a diameter of approximately 2.8 nm (<6.6 nm) in B. megaterium. In contrast, the peptide perturbed the membrane of CHO-K1 cells, permitting the entry of large molecules (diameter, >23 nm) into the cytosol. 

Magainin 2 and PGLa bind preferentially to negatively charged membranes and cause their disruption by pore formation. All-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture produced pore structures (Pino-Angeles et al. 2016). The peptides remained mostly helical and adopted tilted orientations. The calculated tilt angles for PGLa were in agreement with solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembled previously determined NMR and crystal structures. A larger and more ordered pore was seen in the 1:1 heterotetramer with an antiparallel helix arrangement. 

Magainin 2 (MAG2) and PGLa, in the skin of the African frog Xenopus laevis, act by permeabilizing bacterial membranes and exhibit synergism. The amphiphilic MAG2 helix lies flat on the membrane surface in some lipid membranes, but with a tilt angle close to 90 degrees (Strandberg et al. 2016).  In the presence of an equimolar amount of PGLa, MAG2 becomes tilted at an angle of 120 degrees, and its azimuthal rotation angle changes. Since this interaction was found to occur in a concentration range where the peptides per se do not interact with their own type, it seems that MAG2 forms a stable heterodimer with PGLa. Given that PGLa molecules in the complex are known to be flipped into a fully upright orientation, with a helix tilt close to 180 degrees , they must make up the actual transmembrane pore (Strandberg et al. 2016). Possibly the two negative charges on the C-terminus of the obliquely tilted MAG2 peptides neutralize some of the cationic groups on the upright PGLa helices. This would stabilize the assembly of PGLa into a toroidal pore with an overall reduced charge density, which could explain the mechanism of synergy.

Peptides, indolicidin, aurein 1.2, magainin II, cecropin A and LL-37 all cause a general acceleration of essential lipid transport processes without altering the overall structure of the lipid membranes or creating organized pore-like structures (Nielsen et al. 2020). Rapid scrambling of the lipid composition associated with enhanced lipid transport may trigger lethal signaling processes and enhance ion transport.

The generalized transport reaction catalyzed by channel-forming amphipathic peptides is:

Small solutes, electrolytes and water (inNielsen et al. 2020). Rapid scrambling of the lipid composition associated with enhanced lipid transport may trigger lethal signaling processes and enhance ion transport.

The generalized transport reaction catalyzed by channel-forming amphipathic peptides is:

Small solutes, electrolytes and water (in) small solutes, electrolytes and water (out)

References associated with 1.C.16 family:

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Bechinger, B., M. Zasloff, and S.J. Opella. (1993). Structure and orientation of the antibiotic peptide magainin in membrane by solid-state nuclear magnetic resonance spectroscopy. Prot. Sci. 2: 2077-2084. 8298457
Han, M., Y. Mei, H. Khant, and S.J. Ludtke. (2009). Characterization of antibiotic peptide pores using cryo-EM and comparison to neutron scattering. Biophys. J. 97: 164-172. 19580754
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Pino-Angeles, A., J.M. Leveritt, 3rd, and T. Lazaridis. (2016). Pore Structure and Synergy in Antimicrobial Peptides of the Magainin Family. PLoS Comput Biol 12: e1004570. 26727376
Rocha-Roa, C., J.D. Orjuela, C. Leidy, P. Cossio, and C. Aponte-Santamaría. (2021). Cardiolipin prevents pore formation in phosphatidylglycerol bacterial membrane models. FEBS Lett. 595: 2701-2714. 34633077
Strandberg, E., D. Horn, S. Reißer, J. Zerweck, P. Wadhwani, and A.S. Ulrich. (2016). (2)H-NMR and MD Simulations Reveal Membrane-Bound Conformation of Magainin 2 and Its Synergy with PGLa. Biophys. J. 111: 2149-2161. 27851939
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