Abstract
BackgroundDefensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis.ResultsMD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein.ConclusionThe study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.
Highlights
Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants
Many previous studies hypothesized that the major factor defining the antimicrobial activity of defensin is its cationicity or its isoelectric point value, than its secondary structure [5]
The C-terminal region of wild type peptide was more flexible compared to the mutants (Figure 3b)
Summary
Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. The most distinct molecular feature of defensins is their high pI value, ranging from +6 to +12 as monomers, manifested by abundant arginine and lysine residues in their sequences [2] They kill microorganisms through permeabilization of the microbial membrane composed of negatively charged components such as phospholipids, teichoic acids and lipopolysaccharides [3]. Many previous studies hypothesized that the major factor defining the antimicrobial activity of defensin is its cationicity or its isoelectric point (pI) value, than its secondary structure [5]. We made in silico mutants of Apl_AvBD2 with higher pI values than the wild type, and analyzed them by Molecular Dynamic (MD) simulation analysis to find the structure-function relationship. Our results indicated that subtle structural differences in critical areas of the molecule can drastically alter the antibacterial potential of b-defensin molecules
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
