Abstract
Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is an important element against permeability of bactericidal agents, including antimicrobial peptides. However, structural determinants of antimicrobial peptides for LPS recognition are not clearly understood. Pardaxins (Pa1, Pa2, Pa3, and Pa4) are a group of pore-forming bactericidal peptides found in the mucous glands of sole fishes. Despite having a low net positive charge, pardaxins contain a broad spectrum of antibacterial activities. To elucidate the structural basis of LPS interactions of pardaxins, herein, we report the first three-dimensional structure of Pa4 bound to LPS micelles. The binding kinetics of Pa4 with LPS is estimated using [(15)N-Leu-19] relaxation dispersion NMR experiments. LPS/Pa4 interactions are further characterized by a number of biophysical methods, including isothermal titration calorimetry, (31)P NMR, saturation transfer difference NMR, dynamic light scattering, and IR spectroscopy. In the LPS-Pa4 complex, Pa4 adopts a unique helix-turn-helix conformation resembling a "horseshoe." Interestingly, the LPS-bound structure of Pa4 shows striking differences with the structures determined in lipid micelles or organic solvents. Saturation transfer difference NMR identifies residues of Pa4 that are intimately associated with LPS micelles. Collectively, our results provide mechanistic insights into the outer membrane permeabilization by pardaxin.
Highlights
Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is an important element against permeability of bactericidal agents, including antimicrobial peptides
LPS/Pa4 interactions are further characterized by a number of biophysical methods, including isothermal titration calorimetry, 31P NMR, saturation transfer difference NMR, dynamic light scattering, and IR spectroscopy
Reagents—LPS of Escherichia coli 0111:B4 and fluorescein isothiocyanate (FITC)-conjugated LPS from E. coli 055:B5 were purchased from Sigma
Summary
Reagents—LPS of Escherichia coli 0111:B4 and fluorescein isothiocyanate (FITC)-conjugated LPS from E. coli 055:B5 were purchased from Sigma. Two-dimensional TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) spectra of Pa4 in free solution were acquired in an aqueous solution containing 10% D2O at pH 4.5 with 0.5 mM peptide concentration and mixing times of 80 ms and 150 ms, respectively. For the one-dimensional STD experiments, LPS was saturated at Ϫ2.0 ppm (40.0 ppm for reference spectra) with a cascade of 40 selective Gaussian-shaped pulses (49 ms each) in an interval of 1 ms resulting in total saturation time of 2 s. Relaxation Dispersion Measurements—The binding kinetics parameters of Pa4 [15N-Leu-19] to LPS (molar ratio of [LPS]: [Pa4] ϭ 1:12.5) were obtained from two dimensional 1H-15N relaxation dispersion measurements using the Carr-PurcellMeiboom-Gill (CPMG) pulse sequence [48, 49] on a Bruker DRX 700 spectrometer, equipped with an actively shielded cryo-probe and pulse field gradients. Non-polarized spectra were obtained from the parallel (ʈ) and perpendicularly (Ќ) attenuated total reflection polarized spectra, using the expression, 1(ʈ) ϩ 1.44(Ќ), as described previously [56]
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