Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity

Abstract

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.

FIG. 1.

Calcein leakage experiments for Pac-525 in POPG (▵), POPC/POPG (3:1) (○), and POPC (▾) LUVs at 25°C.

FIG.2.

Titration calorimetry of Pac-525 with POPG LUVs (A) and POPC LUVs (B) in PBS buffer (pH 7.4) at 25°C.

FIG. 3.

(A) Fluorescence emission spectra of 1 μM Pac-525 in PBS buffer (□), SDS (•), POPG LUVs (▵), and POPC LUVs (▾) at 25°C. (B) Stern-Volmer plots of 1 μM Pac-525 in PBS buffer (□), SDS (•), POPG LUVs (▵), and POPC LUVs (▾) at 25°C.

FIG. 4.

(A) A 600-MHz TOCSY spectrum, recorded at 70 ms of mixing time of Pac-525 (2 mM) in 200 mM SDS at 37°C, showing the NH (F2)-aliphatic (F1) region. (B) NH-CαH region of the 600-MHz NOESY spectrum recorded at 120 ms of mixing time for Pac-525 (2 mM) in 200 mM SDS at 37°C. Peaks are labeled at the positions of the NH-CαH cross-peaks.

FIG. 5.

Calculated structures of Pac-525. (A) Twenty lowest-energy structures calculated for Pac-525 (2 mM in 20 mM sodium phosphate buffer at pH 4.5 and 37°C with 200 mM SDS). (B) The final refined average structure for Pac-525 bound to SDS. The positively charged residues are shown in blue, the tryptophan residues are shown in yellow, and the hydrophobic residues are shown in gray. (C) The electrostatic surface plot of Pac-525. Positive charge is indicated by blue and neutral charge is indicated by white. The figure was generated using the program MOLMOL (21).