Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

Abstract

The antimicrobial activity of amphipathic ␣-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membranebound state is sparse. Solid state 2 H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentrationdependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125°was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This 2 H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d 3 substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158°is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an ␣-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.

Investigations of the synergistic enhancement of antimicrobial activity in mixtures of magainin 2 and PGLa

“…In particular in thin membranes the peptides exhibits an inherent tendency to adopt transmembrane tilt angles, an effect that is strongly enhanced in the presence of magainin 2 [37,38]. Here we extended these investigations by studying the PGLa L18W variant that has been used previously in studies of antimicrobial action and synergism [34].…”

“…For example, using 15 N solid-state NMR spectroscopy the membrane alignment of PGLa and magainin 2 was monitored. In phosphatidylcholine membranes, where both fatty acyl are saturated, the tendency of PGLa to adopt transmembrane alignments increased in particular in the presence of magainin 2, when at the same time the latter remains surface oriented [37,38]. Importantly, both peptides maintain orientations parallel to the membrane surface in bilayers made of (partially) unsaturated phospholipids (e.g.…”

AMPs and OMPs: Is the folding and bilayer insertion of β-stranded outer membrane proteins governed by the same biophysical principles as for α-helical antimicrobial peptides?

“…We had found this behavior by 2 H-NMR, using Ala-d 3 labeled PGLa in the presence of MAG2 in DMPC/ DMPG (3:1) lipid bilayers[58]. Such an I-state was never observed for PGLa on its own, not even at high peptide concentration [53].…”

“…In all these cases, it is of interest to obtain informa- 58 tion on the structure, orientation and dynamics of the peptide in a The morphology of a lipid assembly in water is determined by the 188 shape of the lipid molecules, when the head groups are exposed to the 189 aqueous solution and the lipid chains are buried inside the assembly. 190 The lipid shape can be described by the number S = V/A o L c , where V 191 is the volume of the hydrocarbon chains of the lipid, A o is the optimal 192 area per lipid, and L c the critical chain length [33].…”

Functional Synergy between Antimicrobial Peptoids and Peptides against Gram-Negative Bacteria

“…One potential solution to the problems of both resistance and toxicity is to use a synergistic combination of antimicrobial compounds, an approach that is ubiquitous in anticancer therapy and is receiving increasing attention in the treatment of infectious diseases (2). Many species produce AMPs with known synergistic interactions, including bacteria (14), insects (20), amphibians (16,31), and humans (3,27,28), with synergy arising through a variety of mechanisms (2,14,29).…”