Homology modelling of wild type and in silico mutated Apl_AvBD2 peptides
The predicted amino acid sequence of Apl_AvBD2 [GenBank: AY641439] was subjected to a homology search using BLAST ([8] and PSI-BLAST [9] against NCBI PDB database. The top hits were aligned against the query sequence in a multiple sequence alignment using Clustal W [10]. We chose the top scoring sequence Apa_AvBD2 (Spheniscin 2: showed 35% sequence identity), originating from King Penguin as the template for further study and the PDB co-ordinate ((PDB id: 1ut3) was retrieved from Protein Data Bank. Homology modelling was carried out using MODELLER package [11] based on the sequence alignment generated between template and target sequences. The atomic coordinates were obtained from the template structures to model Apl_AvBD2. Care was taken to make the coordination geometry of side chain atoms most favorable. Conformations of a few residues were therefore adjusted using loop refinement programme within MODELLER package. Energy minimization of the top scored model was carried out with GROMACS 3.3.1 (The Groningen Machine for Chemical Simulations) [12] using OPLSAA force field. The minimization was set to run for 5000 steps or until convergence to machine precision. PROSA2003 [13] program was used for validation of the model, by analyzing residue interaction energy and z-score. These procedures were iterated several times until a good quality model was obtained.
The dimeric state of Apl_AvBD2 protein was generated using SymmDock [14], an algorithm for the prediction of complexes with symmetry by geometry based docking. The structural representations of Apl_AvBD2 monomers and dimers were analyzed and visualized using PyMol software [15]. Based on the structure of a dimeric complex of Apl_AvBD2 created by the software, the homology models of the in silico mutants were made. Less cationic residues in several positions of the wild type protein were initially mutated to more cationic arginine residues. However, it was found that many of them changed the predicted structure of the protein. So only those mutants, which had the native predicted structure (Figure 1), were selected for further analysis.
Molecular dynamics (MD) simulation of Apl_AvBD2 and Its mutant peptides
Molecular dynamics (MD) simulations were performed using the GROMACS version 3.3.1 and OPLSAA force field. The initial structures were solvated with three-point transferable intermolecular potential (TIP3P) water molecules [16] and appropriate number of Chloride ions in a rectangular box to neutralize the system; the box dimensions ensured that any protein atom was at least 8 Å away from the wall of the box. After energy minimization, MD simulations were performed for 100 ps at constant temperature (300 K) and pressure (1 atm) with periodic boundary conditions, particle-mesh Ewald summation, and a 1-fs time step to heat and equilibrate the system. This was followed by production runs of 10 ns duration for each simulation. Structures were saved every 10 ps for analysis. The relative binding energies computed using the tool g_energy module of 'GROMACS 3.3.1, employing molecular mechanics and a continuum solvent model. The output files (.xvg) from the GROMACS 3.3.1 was analysed in XMGRACE [17] software.
The parameters analysed were: area per atom, area per residue, energy variations (kinetic energy, potential energy and total energy), van der Waals interactions, intra-molecular hydrogen bonds, inter-molecular hydrogen bonds with surrounding water molecules, radius of gyration, root mean square deviations (RMSD) of each amino acid residue, root mean square fluctuations (RMSF) of Cα atoms of each amino acid residue, solvent accessible surface area, and hydrophobic and hydrophilic interactions. Pressure, volume, temperature, minimum distance to periodic image and maximum internal distance of the MD simulation system were examined. The obtained parameters for wild type and mutant peptides were compared.
In vitro site directed mutagenesis and evaluation of antibacterial activity of wild type and mutant Apl_AvBD2
Mutagenesis reactions were carried out using the modified mutagenic primers as per the protocol by Quick Change Mutagenesis Kit (Stratagene, La Jolla, CA). Apl_AvBD2-pET-32 gene-vector construct was used for this purpose. The cloning strategy and recombinant expression protocols are described elsewhere [6]. Selected clones were sequenced directly using an ABI 3730 Genetic Analyzer automated DNA sequencer (PE Applied Biosystems, Foster City, CA) for confirmation of mutation. Recombinant proteins of single amino acid mutants and serial progressive mutants of Apl_AvBD2, which were selected for the MD analysis (Figure 1), were made in vitro. These would make variants of the protein with pI value ranging from 8.69 to 9.97. These proteins were expressed in BL21DE3 pLysS bacterial cells, purified and used for antibacterial assay [6].