Structural insights into the combinatorial effects of antimicrobial peptides reveal a role of aromatic-aromatic interactions in antibacterial synergism

Abstract

The recent development of plants that overexpress antimicrobial peptides (AMPs) provides opportunities for controlling plant diseases. Because plants employ a broad-spectrum antimicrobial defense, including those based on AMPs, transgenic modification for AMP overexpression represents a potential way to utilize a defense system already present in plants. Herein, using an array of techniques and approaches, we report on VG16KRKP and KYE28, two antimicrobial peptides, which in combination exhibit synergistic antimicrobial effects against plant pathogens and are resistant against plant proteases. Investigating the structural origin of these synergistic antimicrobial effects with NMR spectroscopy of the complex formed between these two peptides and their mutated analogs, we demonstrate the formation of an unusual peptide complex, characterized by the formation of a bulky hydrophobic hub, stabilized by aromatic zippers. Using three-dimensional structure analyses of the complex in bacterial outer and inner membrane components and when bound to lipopolysaccharide (LPS) or bacterial membrane mimics, we found that this structure is key for elevating antimicrobial potency of the peptide combination. We conclude that the synergistic antimicrobial effects of VG16KRKP and KYE28 arise from the formation of a well-defined amphiphilic dimer in the presence of LPS and also in the cytoplasmic bacterial membrane environment. Together, these findings highlight a new application of solution NMR spectroscopy to solve complex structures to study peptide-peptide interactions, and they underscore the importance of structural insights for elucidating the antimicrobial effects of AMP mixtures.

Keywords: antibiotic resistance; antimicrobial peptide (AMP); lipopolysaccharide (LPS); nuclear magnetic resonance (NMR); peptide conformation; synergism; transferred NOESY.

Figure 1.

A, primary amino acid sequences of the parent peptides, VG16KRKP and KYE28, as well as of their mutant analogs. The three-dimensional structure of the individual peptides in LPS is shown on right. The boldface amino acid residues are the mutated ones. B, checkerboard analysis using a modified micro broth dilution assay was performed to calculate the values of MIC_99% and FICI. As shown, the parent VG16KRKP and KYE28 peptides interact with each other, demonstrated by the lowering of individual peptide concentration required for microbial killing. All experiments were performed in triplicate. C, proteolytic stability of the respective parent peptides, VG16KRKP and KYE28, in tomato plant extract containing plant proteases was evaluated using HPLC-based analysis. Both peptides displayed a half-life greater than 120 min. The peptides were incubated individually with the plant extract at 310 K, and aliquots were drawn at regular intervals and analyzed using RP-HPLC.

Figure 2.

A, SEM images of X. vesicatoria in the absence of both the peptides VG16KRKP and KYE28 (control) showed no visible morphological changes after 1 h of incubation. In contrast, cells exposed to 0.5 × MIC_99% (MIC_99% = 1 μm each) showed onset of cell distortion (B), and exposure at 1× MIC_99% resulted in pronounced membrane perturbation with extensive release of intracellular constituents and loss of cellular morphology (C). The scale corresponds to 10 μm. Additionally, dye-based fluorescence assay with live X. vesicatoria cells displayed that the peptide combination at 1× and 2× MIC_99% concentration could effectively interact with bacterial cell membrane bringing about a change in membrane potential as indicated by an increase in DISC₃ fluorescence (D), thus disrupting the outer membrane comprising of lipopolysaccharide (E and F), and the inner membrane made of negatively charged lipid components (G). The control cell showed baseline fluorescence under all conditions. It should be noted that ANS and NPN are lipophilic dyes, which fluoresces strongly in nonpolar environments of the cell membrane, which gets exposed upon membrane disintegration. PI, however, binds to nucleic acid components of the dead cell conforming inner membrane disruption. Each experiment was recorded for 30 min at 298 K.

Figure 3.

A, dynamic light-scattering experiment was performed with monodisperse population of spherical 3:1 POPE/POPG LUV. The concentrations stated indicate equimolar absolute concentrations of the two peptides individually, in μm. Upon titration with VG16KRKP and KYE28 mixtures, the vesicles showed significant changes in shape and size, as indicated by the increased PDI value and the increase in delay time (black broken line), respectively (left side). Each experimental set was done in triplicate. B, ³¹P solid-state NMR studies further supported VG16KRKP–KYE28–induced vesicle deformation and aggregation (right) as depicted by the change in intensity of the parallel (∼30 ppm) and perpendicular (∼−15 ppm) edges and an increased spectral span. The inset shows immediate vesicle flocculation upon addition of the peptides, whereas the control vesicles remain unchanged. In contrast, as seen on the left side, the peptide combination could only cause flocculation but no morphological changes in the bicelles prepared from E. coli total lipid extract bicelle.

Figure 4.

A, two-dimensional ¹H-¹H trNOESY spectra of VG16KRKP–KYE28 complex in the LPS micelle, displaying the NOE contacts in the fingerprint region for C^αH-NH and NH-NH resonances important for structure stabilization. B, parent peptide complex also displayed intra- and inter-molecular long-range NOE contacts, illustrating the importance of aromatic residues in the formation of a stabilized structure and justifying the strong synergistic interaction observed. The experiments were performed by titrating 0.6 mm of the total peptides with 10 μm LPS (LPS: each peptide = 1:30) at pH 4.5, using a Bruker Avance III 700 MHz NMR spectrometer (NOESY mixing time = 150 ms) and at 298 K (residues marked in blue are from VG16KKRP and in red are from KYE28).

Figure 5.

A, two-dimensional ¹H-¹H trNOESY spectra of VG16KRKP–KYE28 in a 3:1 POPE/POPG vesicle, displaying the NOE contacts in the fingerprint region for C^αH-NH and NH-NH resonances important for structure stabilization. VG16KRKP–KYE28 upon interaction with 3:1 POPE/POPG vesicles exhibited several trNOEs hinting toward the adoption of a rigid and well-converged structure. B, parent peptide complex, also displayed intra- and inter-molecular long-range NOE contacts. The experiments were performed by titrating 0.6 mm of the total peptides with 12 μm of 3:1 POPE/POPG LUV (LUV: each peptide = 1:25) at pH 4.5, using a Bruker Avance III 700 MHz NMR spectrometer (NOESY mixing time = 150 ms) and at 298 K (residues marked in blue are from VG16KKRP and in red from KYE28).

Figure 6.

A and B, 15 ensemble LPS-bound structure of the VG16KRKP–KYE28 complex showed a well-defined conformation with good convergence of the backbone atoms (Protein Data Bank code 6KBO). C, cartoon representation of the complex indicated adoption of an amphipathic structure stabilized by an inter-molecular aromatic–aromatic hub, with the agreement of an overall “helix-loop-helix” structure. Interestingly, both the peptides showed a marked difference from the original structure in LPS, which could give a plausible justification for their enhanced activity. D, hydrophobic hub of VG16KRKP–KYE28 complex was characterized by energetically favorable cluster of aromatic–aromatic interactions, especially T-shaped geometries, formed between aromatic residues, which maintained a spatial distribution of ∼3.5–7 Å between them. E, two positively charged clusters with an end-to-end distance of 29 Å, corroborating closely with the thickness of the LPS bilayer, giving subtle hints toward the mode of action of the peptide complex. F, electrostatic surface potential of the LPS-bound complex at an angle of 180° further depicted the clear demarcation of the surface charge distribution, generated using MOLMOL software.

Figure 7.

A and B, 15 ensemble 3:1 POPE/POPG LUV-bound structure of the VG16KRKP–KYE28 complex displayed a well-defined conformation that, when compared with the LPS-bound structure, was more flexible (Protein Data Bank code 6KBV). C and D, one molecule cartoon representation of the complex indicated adoption of an amphipathic “helix-loop-helix” structure with a clear separation of charge with the hydrophobic network formed by the bulky aromatic groups as well as aliphatic residues involved in intra- and inter-molecular aromatic–aromatic and aromatic–aliphatic interaction via three T-shaped interactions between the residues Phe-12^VG16KRKP–Trp-5^VG16KRKP–Phe-23^KYE28, Trp-5^VG16KRKP–Tyr-2^KYE28–Phe-12^VG16KRKP, and Phe-19^KYE28–Trp-5^VG16KRKP–Phe-23^KYE28, whereas E, the cationic face remained exposed to the solvent front, forming a single tight cluster of positive charges, which aids in initial electrostatic interaction with the negatively charged phosphate headgroups of the lipid molecules. F, electrostatic surface potential of the vesicle-bound complex at an angle of 180° further depicted the clear demarcation of the surface charge distribution, generated using MOLMOL software.

Figure 8.

A, PRE NMR was performed using MTSL-tagged VG16KRKP with KYE28 in LPS. The trNOESY experiment was conducted under similar conditions with the unlabeled peptide to compare residue-specific T₂ relaxation, depicted as I/I₀. The residues in the vicinity of the labeled Cys-9 residue exhibited almost complete relaxation as shown by broadened Hα/NH trNOESY peaks. B, similarly, H/D experiments performed throughout 5 h corroborated with the solved structure with residues involved in the formation of the hydrophobic hub and those engaged in π-cation interaction remaining shielded from the exchange kinetics. C, FRET experiment showed a linear change in the emission intensity of the Trp residue of the donor peptide, VG16KRKP upon titration with increasing concentrations of dansylated acceptor KYE28, upto 0.5 mol fraction. The FRET experiment was performed at 298 K in 10 mm potassium phosphate buffer (pH 7.4).