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.