Mechanism Analysis of Antimicrobial Peptide NoPv1 Related to Potato Late Blight through a Computer-Aided Study

Abstract

Phytophthora infestans (Mont.) de Bary, the oomycotic pathogen responsible for potato late blight, is the most devastating disease of potato production. The primary pesticides used to control oomycosis are phenyl amide fungicides, which cause environmental pollution and toxic residues harmful to both human and animal health. To address this, an antimicrobial peptide, NoPv1, has been screened to target Plasmopara viticola cellulose synthase 2 (PvCesA2) to inhibit the growth of Phytophthora infestans (P. infestans). In this study, we employed AlphaFold2 to predict the three-dimensional structure of PvCesA2 along with NoPv peptides. Subsequently, utilizing computational methods, we dissected the interaction mechanism between PvCesA2 and these peptides. Based on this analysis, we performed a saturation mutation of NoPv1 and successfully obtained the double mutants DP1 and DP2 with a higher affinity for PvCesA2. Meanwhile, dynamics simulations revealed that both DP1 and DP2 utilize a mechanism akin to the barrel-stave model for penetrating the cell membrane. Furthermore, the predicted results showed that the antimicrobial activity of DP1 was superior to that of NoPv1 without being toxic to human cells. These findings may offer insights for advancing the development of eco-friendly pesticides targeting various oomycete diseases, including late blight.

Keywords: Phytophthora infestans; biopesticide; dynamic simulation; molecular docking; potato.

Figure 1

PvCesA2 homology models in detail. (A) pLDDT per position. (B) Measurement of model confidence. This section presents the quality of the model alongside its corresponding pLDDT score. (C) The predicted aligned error (PAE) is visually represented for each structural prediction.

Figure 2

Helix-wheel analysis of the peptides. Polar neutral amino acids are shown in yellow; Basic residues are shown in blue; Acidic residues are shown in red; and Non-polar amino acids are shown in grey. The peptide’s head is denoted by the red letter “N”, whereas its tail is marked by the red letter “C”.

Figure 3

Electrostatic surface and the binding sites of NoPvs docked in PvCesA2. A. NoPv1, NoPv1R1A, and NoPv1R1AR7A bind on site A; B. NoPv2, NoPv3, and NoPv1R7A bind on site B. NoPv1 is shown in red, NoPv1R1A is shown in green, NoPv1R1AR7A is shown in cyan, NoPv2 is shown in yellow, NoPv3 is shown in orange, and NoPv1R7A is shown in purple. Blue surfaces represent electropositive surfaces, while red surfaces represent electronegative surfaces.

Figure 4

Interactions of the NoPv peptides with PvCesA2. (A) Interaction between NoPv1 and PvCesA2. (B) Interaction between NoPv1R1A and PvCesA2. (C) Interaction between NoPvR1AR7A and PvCesA2. (D) Interaction between NoPv1R7A and PvCesA2. (E) Interaction between NoPv2 and PvCesA2. (F) Interaction between NoPv3 and PvCesA2. The green dashed lines represent conventional hydrogen bonds.

Figure 5

Predicted mutation energy of mutants for PvCesA2-NoPv1 complexes at pH 7.4.

Figure 6

The electrostatic potential on the surface of peptides. Blue surfaces represent electropositive surfaces, while red surfaces represent electronegative surfaces.

Figure 7

Electrostatic surface rendering of the PvCesA2-peptide complexes during the MD simulation. (A) NoPv1-PvCesA2. (B) DP1-PvCesA2. (C) DP2-PvCesA2. Blue surfaces represent electropositive surfaces, while red surfaces represent electronegative surfaces. L0, L30, L60, L90, L120, L150, L180, L210, L240, L270, and L300 are colored in cyan, carbon, dash, blue, pink, forest, firebrick, orange, olive, purple, and gray. (e.g., L30 represents the location of NoPv1/DP1/DP2 at 30 ns during the MD simulation period).

Figure 8

The FMOs including HOMO and LUMO for (A) NoPv1, (B) DP1, (C) DP2, as calculated at the B3LYP-D3/6-31+G(d,p) level of DFT. The positive and negative phases of the orbital wave function are shown in light green and light blue respectively.

Figure 9

Translocation process diagram of the DP1, with the N-terminus approaching the surface of the cell membrane. (A) 0 ps. (B) 200 ps. (C) 600 ps. (D) 900 ps. (E) 1300 ps. (F) 1500 ps.

Figure 10

Schematic illustration of DP1 translocation process at (A) 200 ps; (B) 600 ps; (C) 900 ps; (D) 1300 ps. Non-polar amino acids are shown in orange. Polar amino acids are shown in cyan. The phospholipid molecules are shown in green.

Figure 11

Translocation process diagram of DP1, with the N-terminus approaching the surface of the cell membrane. (A) 0 ps. (B) 300 ps. (C) 600 ps. (D) 900 ps. (E) 1300 ps. (F) 1600 ps.

Figure 12

Schematic illustration of the DP2 translocation process at (A) 300 ps; (B) 600 ps; (C) 900 ps; (D) 1300 ps. Non-polar amino acids are shown in orange. Polar amino acids are shown in cyan. The phospholipid molecules are shown in green.