The solution structure of a novel plant defensin (PhD1) contains a fifth disulfide bond, unlike other plant defensins, which have four disulfide bonds. The present study aims to better understand the stability, thermal dependence and the role of disulfide bonds in the tertiary structure of PhD1 using Molecular Dynamics (MD) simulations. The secondary structures are intact in the native structure simulated at 300 K. However, in the mutant structures a small variation is observed. A significant shift in the peptide conformation is observed when the additional fifth disulfide bond (Cys7-Cys23) is mutated. No large change in the tertiary structure conformation is observed till 400 K, which demonstrates the high thermal stability of the protein. Here, we also show that the mutation of disulfide bonds did not result in a drastic conformational change. The antifungal property along with high structural stability of the plant defensin protein makes it a promising candidate for the development of novel fungicides.

Abstract:The three-dimensional structure of a cysteine rich antifungal protein EAFP2 is found to be compact and extremely stable. The rigid nature of the protein is attributed to the presence of five disulfide bonds. However, the effect of individual disulfide bonds on the conformation has not been characterized. Thus, Molecular Dynamics (MD) simulations are used to explicate the influence of disulfide bonds on the conformation. In the present study, the cysteine residues in the native structure are mutated to alanine and the structural characteristics and conformational dynamics of the native and mutant structures are analyzed to better understand the effect of disulfide bonds on the tertiary structure. The simulated native and single mutant structures are found to posses similar conformations, indicating that loss of disulfide bond did not affect the tertiary structure conformation greatly. However, in the single mutant (C7A) structure, the N and C-terminal segments are found to be different. It is also interesting to note that the loss of disulfide bond between Cys35 and Cys39 actually resulted in a more compact structure.