Work

Functional transformation of leucine-rich repeat (LRR) polygalacturonase inhibitor protein (PGIP) of Moringa oleifera by dual mutation E146G and E218K in defense against Cercospora baticola

Year: 2025

Type: article

Abstract: Cercospora baticola, a fungal phytopathogen, is responsible for causing brown spot disease in Moringa oleifera by secreting polygalacturonases (PGs), a class of enzymes that degrade pectin in the plant cell wall to facilitate infection. In response, M. oleifera mounts a defense mechanism by producing polygalacturonase-inhibiting proteins (moPGIPs), which act as inhibitors by forming direct protein–protein interactions with fungal PGs, thereby neutralizing their enzymatic activity.This study aims to elucidate the structural basis of the moPGIP–cbPG interaction and to evaluate the functional consequences of two specific site-directed mutations E146G and E218K located within the conserved leucine-rich repeat (LRR) domains of moPGIP. These mutations were designed to explore their impact on the protein’s ability to bind and inhibit cbPG. Protein structures were predicted using multiple homology modeling platforms, and site-directed mutations were introduced using PyMOL. Structural validations were performed through Ramachandran plot analysis and energy scoring. Rigid docking between moPGIP (wild-type and mutants) and cbPG was conducted using HDOCK, followed by 100 ns molecular dynamics (MD) simulations to evaluate interaction stability and dynamic behavior. Comparative analysis between the wild-type and double-mutant moPGIP revealed several key differences. The mutant complex exhibited weakened binding affinity (−0.57 kcal/mol vs. −0.66 kcal/mol), increased residue-level flexibility as indicated by root-mean-square fluctuations (RMSF), greater conformational diversity (principal component trace: 115.9 nm² vs. 48.4 nm²), and decreased structural compactness, reflected by a broader radius of gyration (Rg: 2.8–6.0 nm). Additionally, solvent-accessible surface area (SASA) and atomic density measurements indicated a destabilized interaction interface in the mutant complex. Principal component analysis (PCA) and free energy landscape (FEL) mapping further confirmed enhanced conformational flexibility and energetic heterogeneity in the mutant structure.Collectively, these results provide detailed molecular insights into PGIP-mediated resistance mechanisms in M. oleifera and highlight the functional relevance of conserved residues in maintaining stable PG–PGIP interactions. This study not only advances the understanding of host–pathogen molecular interfaces but also offers a robust computational framework for guiding protein engineering strategies aimed at enhancing disease resistance in Moringa.

Source: The Microbe

DOI: 10.1016/j.microb.2025.100519