High-Resolution Structures and Orientations of Antimicrobial Peptides Piscidin 1 and Piscidin 3 in Fluid Bilayers Reveal Tilting, Kinking, and Bilayer Immersion

Abstract

While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of 1H–15N dipolar couplings and 15N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (∼25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ∼1.2–3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.

Simulations of Membrane-Disrupting Peptides II: AMP Piscidin 1 Favors Surface Defects over Pores

“…In four of the 20 peptides, the first three residues frayed supporting the previous observation that the N-terminal residues are the most disordered (30). This fraying is more pronounced for TM peptides, which were observed to transiently unravel their first three N-terminal residues to partition the phenylalanines in the hydrocarbon core before reaching the interface and refolding into an a-helical secondary structure.…”

“…As already noted, p1 contains a kink. This kink, which is formed by a relative rotation of the a-helical segments on either side of the central glycine 13 (termed Dr), does not bend the helix and has been proposed to stabilize the S state by maximizing the hydrophobic moment of the helix (30). As shown in Fig.…”

“…Like other AMPs, piscidin induces dye leakage in model membranes and membrane disruption is proposed to be its primary mechanism of action against bacteria (28,29). The surfacebound structures of p1 were determined independently by ssNMR and all-atom molecular dynamics (MD) in two different bacterial cell mimics (30). Both methods yielded highly helical structures parallel to the membrane surface, and a [20][21][22][23][24][25][26][27][28][29][30] kink about the helical axis.…”

“…The surfacebound structures of p1 were determined independently by ssNMR and all-atom molecular dynamics (MD) in two different bacterial cell mimics (30). Both methods yielded highly helical structures parallel to the membrane surface, and a [20][21][22][23][24][25][26][27][28][29][30] kink about the helical axis. Subsequent MD simulations demonstrated membrane thinning and induction of positive spontaneous curvature by p1 in 3:1 POPC/POPG bilayers (31).…”

“…The first two subsections of the Results and Discussion report a 26 ms trajectory of 20 transmembrane p1 in four pores, and a 2.5 ms trajectory initialized from 16 surfacebound p1; both simulations were carried out in 3:1 POPC/ POPG and a peptide/lipid ratio of 1:20 (the same as previous ssNMR measurements (30)) at 313 K with no external electric field. Peptide and lipid orientations in the simulations are then compared to orientations measured by ssNMR at three different temperatures (278, 285, and 305 K).…”