Analysis of mutations of defensin protein using accelerated molecular dynamics simulations.

Abeer Hashem,Elsayed Fathi Abd_Allah,Ayyalusamy Ramamoorthy,Awdhesh Kumar Mishra,Chetna Tyagi,Tapan Kumar Mohanta,Abdulaziz A Alqarawi,Gopal Kumar Prajapati,Bharati Pandey

doi:10.1371/journal.pone.0241679

Abstract

Plant defensins possess diverse biological functions that include antifungal and antibacterial activities and α-amylase and trypsin inhibitory properties. Two mutations, G9R and V39R, were confirmed to increase the antifungal activity of Raphanus sativus antifungal protein 2 (RsAFP2). Accelerated Molecular Dynamics (aMD) were carried out to examine the conformational changes present in these RsAFP2 mutants, and its two closest homologs compared to the wild-type protein. Specifically, the root mean square fluctuation values for the eight cysteine amino acids involved in the four disulfide bonds were low in the V39R mutant compared to the wild-type. Additionally, analysis of the free energy change revealed that G9R and V39R mutations exert a neutral and stabilizing effect on RsAFP2 conformation, and this is supported by the observed lower total energy of mutants compared to the wild-type, suggesting that enhanced stability of the mutants. However, MD simulations to a longer time scale would aid in capturing more conformational state of the wild-type and mutants defensin protein. Furthermore, the aMD simulations on fungal mimic membranes with RsAFP2 and its mutants and homologs showed that the mutant proteins caused higher deformation and water diffusion than the native RsAFP2, especially the V39R mutant. The mutant variants seem to interact by specifically targeting the POPC and POPI lipids amongst others. This work highlights the stabilizing effect of mutations at the 9th and 39th positions of RsAFP2 and their increased membrane deformation activity.

Highlights

Plants have evolved natural resistance to abiotic and biotic stresses, such as adverse environmental conditions and infection by microbial and insect pathogens
Plants are exposed to a wide range of pathogenic fungi, and to combat, plants have evolved defense mechanisms that include the use of low molecular weight proteins that possess antifungal activity
Cys25 was found to be substituted to arginine (Fig 1). These findings indicate that these residues are subjected to considerably strong selection pressure, as they play a critical role in the detection of various fungal species

Summary

Introduction

Plants have evolved natural resistance to abiotic and biotic stresses, such as adverse environmental conditions and infection by microbial and insect pathogens. Plant defensins (formerly known as γ-thionins) possesses different structural configuration (β1-αβ2-β3) from mammalian defensins (α-β1-β2-β3) They are small cationic peptides consisting of 45-54 amino acids that comprise four to five intramolecular disulfide bonds [4,5,6]. These defensins exhibit diverse biological functions that include antifungal [7,8,9,10,11,12] and antibacterial [13, 14] activities, and α-amylase and trypsin inhibitory properties [15, 16]. In a recent study [23], demonstrated that peptide chain length and conformation locking are very critical parameters while designing potent novel antimicrobial peptide

Methods

Results

Conclusion