Ll-37-Derived Aurein 1.2 Analog

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain HN protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the epsilon HN protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.

High-resolution two-dimensional (2D) (1)H-(15)N heteronuclear correlation (HETCOR) spectroscopy has been used to characterize the structure and dynamics of (15)N-backbone labeled antimicrobial piscidin 1 (p1) oriented in "native-like" hydrated lipid bilayers. Piscidin belongs to a family of amphipatic cationic antimicrobial peptides, which are membrane-active and have broad spectrum antimicrobial activity on bacteria. When the (1)H chemical shifts are encoded by the (1)H-(15)N dipolar couplings, 2D dipolar-encoded HETCOR (i.e., de-HETCOR) solid-state NMR spectra yield high resolution (1)H and (15)N chemical shifts as well as (1)H-(15)N heteronuclear dipolar couplings. Several advantages of HETCOR and de-HETCOR techniques that emerge from our investigations could facilitate the atomic-level investigations of structure-function relationships in membrane-active peptides and membrane-bound species. First, the de-HETCOR NMR spectrum of a ten-site (15)N-labeled sample of p1 aligned in hydrated lipid bilayers can resolve resonances that are overlapped in the standard HETCOR spectrum. Second, the resolution in de-HETCOR spectra of p1 improves significantly at higher magnetic field due to an enhanced alignment that improves spectrum definition uniformly. Third, the HETCOR spectrum of (15)N-K(14) p1 oriented in hydrated lipid bilayers displays not only the expected crosscorrelation between the chemical shifts of bonded amide(1)H and (15)N spins but also a cross peak between the (1)H chemical shift from bulk water and the (15)N chemical shift from the labeled amide nitrogen. This information provides new insights into the intermolecular interactions of an amphipathic antimicrobial peptide optimized to partition at the water-bilayer interface and may have implications at the biological level.

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA>aurein 1.2>>the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by (1)H NMR spectroscopy, including structural refinement by natural abundance (13)C(alpha), (13)C(beta), and (15)N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 A below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.