4.4.1 Evaluating the binding affinity of CARD-like domain of Rv2615c with CARD-APAF1 and CARD-Caspase9
Molecular docking of Rv2615c protein was performed to estimate the binding affinity of CARD-like domain of Rv2615c with CARD-APAF1 and CARD-Caspase9 using PatchDock server (Schneidman-Duhovny et al. , 2005). Top ten docked structures were further refined in FireDock server (Mashiach et al. , 2008). The best model was evaluated based on number of bonds formed and the energy score generated in HEX 8.0.0. Visualization was done in Discovery studio Visualizer 4.1 (Accelyrs Inc., USA). The complex of CARD-APAF1 with CARD-Caspase9 (PDB ID: 3YGS) was included as positive control and the inactivated independent APAF-ADP complex (PDB ID: 1Z6T) was included as negative control in the study.
We observed Leucine-116 and Isoleucine-117 were two common residues within CARD-like domain of Rv2615c which aligned with conserved residues Methionine-29 and Isoleucine-30 of CARD-APAF1 and Leucine-58 and Isoleucine-59 of CARD-Caspase9. Therefore, site directed mutation was incorporated changing Leucine-116 and Isoleucine-117 to the most preferred Alanine substitution in CARD-like domain of Rv2615c and the mutant structure was checked for its stability using I-Mutant 2.0 server (Capriotti, Fariselli and Casadio, 2005). Furthermore, binding affinity of this mutant Rv2615c protein with CARD-APAF1 and with CARD-Caspase9 was evaluated through docking studies as described previously.