title: Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2.
abstract: BACKGROUND
  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. To test this hypothesis and to improve the activity of the newly identified avian beta-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.
  
  
  RESULTS
  MD 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Molecular Virology Laboratory, Department of Molecular Microbiology, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram-695014, Kerala, India. soja.saghar@gmail.com
authors.forename: Soja Saghar
authors.initials: SS
authors.lastname: Soman
authors.forename: Krishnankutty Chandrika
authors.initials: KC
authors.lastname: Sivakumar
authors.forename: Easwaran
authors.initials: E
authors.lastname: Sreekumar
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides; D030161:Avian Proteins / Q000737:chemistry*; D001717:Birds; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D023083:beta-Defensins / Q000737:chemistry*
chemical_list: D023181:Antimicrobial Cationic Peptides; D030161:Avian Proteins; D011994:Recombinant Proteins; D023083:beta-Defensins