# Screening and Validation of Functional Residues of the Antimicrobial Peptide PpRcys1

## TL;DR

This study identifies key amino acid residues in the antimicrobial peptide PpRcys1 that are essential for its antibacterial activity against bacteria in aquaculture.

## Contribution

The study identifies and validates the functional residues in PpRcys1 that are critical for its antibacterial activity and membrane interaction.

## Key findings

- ARG40, LYS55, LYS90, and LYS93 are critical for PpRcys1's interaction with bacterial membranes.
- Mutating these residues significantly reduced the peptide's antibacterial activity and membrane binding ability.
- The loss of these residues prevented PpRcys1 from compromising bacterial membrane integrity.

## Abstract

The excessive use of conventional antibiotics in aquaculture has created significant challenges, making it essential to explore and develop effective alternatives. Antimicrobial peptides (AMPs) have gained attention as potential therapeutic agents owing to their wide-ranging antibacterial effects and their ability to address pathogens resistant to conventional drugs. PpRcys1 is an antimicrobial peptide that mainly targets bacterial cell membranes, exhibiting a minimum inhibitory concentration of 8–32 μM. Its antibacterial activity should be further optimized. Before such optimization, however, it is crucial to identify the key amino acid residues that determine its functional activity. In this study, molecular dynamics simulations indicated that arginine 40 (ARG40), lysine 55 (LYS55), lysine 90 (LYS90), and lysine 93 (LYS93) play critical roles in the interaction between PpRcys1 and bacterial membranes. To investigate this further, these residues were mutated to serine, producing the mutant peptide PpRcys1_RMRK. Compared with PpRcys1, the mutant peptide PpRcys1_RMRK showed a significant reduction in antibacterial activity. Results from molecular dynamics simulations, Western blot, and ELISA demonstrated a marked decrease in its ability to bind to bacterial cell membranes. Membrane permeation assays, cell membrane depolarization experiments, and scanning electron microscopy revealed that PpRcys1 could not compromise the integrity of the bacterial membrane after losing ARG40, LYS55, LYS90 and LYS93. These findings highlight the critical roles of ARG40, LYS55, LYS90, and LYS93 in sustaining the antibacterial activity of PpRcys1. This study provides important initial insights into the structure–activity relationship of PpRcys1 and establishes a theoretical foundation for its future optimization.