Membrane-Bound Dimer Structure of a β-Hairpin Antimicrobial Peptide from Rotational-Echo Double-Resonance Solid-State NMR

Abstract

The intermolecular packing of a beta-hairpin antimicrobial peptide, PG-1, in lipid bilayers is determined using solid-state NMR distance measurements. Previous spin counting experiments showed that PG-1 associates as dimers in POPC bilayers; however, the detailed dimer structure was unknown. We have now measured several intermolecular 13C-19F, 1H-13C, and 15N-13C distances in site-specifically labeled PG-1 to constrain the structure of the intermolecular interface. The distances are measured using the rotational-echo double-resonance (REDOR) technique under magic-angle spinning. The results indicate that two PG-1 molecules align in a parallel fashion with the C-terminal strand of the hairpin forming the dimer interface. Six hydrogen bonds stabilize this interface, and the Phe12 side chain adopts the g- conformation in the membrane as in solution. The parallel packing of the peptide in the lipid bilayer differs from the antiparallel dimer found in DPC micelles and may be stabilized by its strong amphipathic character, which should facilitate its insertion into the amphipathic lipid bilayer. This study demonstrates the utility of the REDOR NMR technique for the elucidation of the oligomeric structure of membrane proteins.

Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

“…In our hands, higher order AMPs also tend to be more cytotoxic, reflecting their increased interactions with mammalian membranes. B2088 retains its monomeric state in various media, which is in agreement with biophysical observations of other AMPs (51,63,79). The trend in designing AMPs toward higher covalent order might be limited by increased cytotoxicity and can be counteracted by designing lower order AMPs, maintaining high mobility in LPS and providing higher concentrations at the lipid surfaces of bacteria.…”

Membrane-dependent oligomeric structure and pore formation of a β-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR

“…In the anionic membrane, PG-1 contacts both the lipid headgroup and acyl chains and thus is well inserted. The peptide assembles into (NCCN) n multimers whose like-stranded interfaces are constrained not only by F-F distances but also by previously measured heteronuclear distances in POPC bilayers (20), which indicated parallel packed and hydrogen-bonded C strands. Such a hydrogen-bond stabilized (NCCN) n multimer strongly suggests the formation of a ␤-barrel.…”

“…This hypothesis is inconsistent with the current data. Instead, the PG-1 ␤-barrel has tight C strand interfaces, where the shortest intermolecular distance is 2.5 Å between Cys 15 CЈ and Cys 15 H N (20). Even the N strand interfaces, with fewer distance constraints so far, have a remarkably short Phg 7 -Phg 7 distance (6.5 Å) and a high percentage (75%) of dimerization.…”

“…The uncertainties of S͞S 0 , S/S 0 , were propagated from the signal-to-noise ratios (SNR) of the S 0 and S spectra according to S/S 0 ϭ (S͞S 0 )⅐[ S0 2 ϩ S 2 ] 1/2 , where is 1͞SNR (20). Sideband intensities were included in the SNR calculation by using SNR total ϭ SNR 0 ⅐(͚P i 2 ͞P 0 2 ), where P 0 is the intensity of the highest peak of the spectrum.…”

A Two‐Tailed Phosphopeptide Crystallizes to Form a Lamellar Structure

“…Further ssNMR structural study of the MAX1 peptide, which has a very similar structure, showed that such peptides stack in an anti (head‐above‐tail) orientation 8d. Other amphipathic β‐hairpins have formed face‐to‐face dimers both in syn (head‐above‐head) and anti relative orientations, which can be affected by their environment 11. The phosphopeptide bilayer is comparable to that of amphiphilic peptide arrangements of KLVFF‐containing peptides, which form helical bilayer nanotapes that close to form nanotubes 12.…”