2.4.1 CARD-like domain of Rv2615c has strong binding affinity
for CARD domains of APAF1 and Caspase9
Docking studies with whole Rv2615c protein revealed that its CARD-like
domain could bind strongly with CARD-APAF1 (Hex energy score: -655.54
Kcal/mol) and with CARD-Caspase9 (Hex energy score: -694.29 Kcal/mol)
(Fig 6). Rv2615c formed 25 bonds (3 electrostatic, 7 hydrogen and 15
hydrophobic bonds) with CARD-APAF1 while 24 bonds (6 electrostatic, 6
hydrogen and 12 hydrophobic bonds) with CARD-Caspase9 through its
CARD-like domain (Fig S6). These Hex energy scores were much higher than
the Hex scores of positive controls included in the study i.e., complex
of CARD-APAF1 with CARD-Caspase9 (PDB ID 3YGS- Hex score: -463.25
Kcal/mol) (Fig 6). However, Hex score for Rv2615c docked with APAF1-ADP
was very less (-110.48 Kcal/mol) compared to the positive control. This
could possibly be because of low affinity of Rv2615c for inactive form
of APAF1 protein that is not bound to Apoptosome complex.
We observed two crucial adjacent hydrophobic residues i.e., Leucine-116
and Isoleucine-117 in CARD-like domain of Rv2615c which were common,
totally conserved or similar and aligned with conserved residues of
CARD-APAF1 (Methionine-29 and Isoleucine-30) and CARD-Caspase9
(Leucine-58 and Isoleucine-59). These residues (Leucine-116 and
Isoleucine-117) within Rv2615c were also involved in bond formation with
CARD-APAF1 and CARD-Caspase9 (Fig S6). Mutant of Rv2615c in which
Leucine-116 and Isoleucine-117 within the CARD-like domain were mutated
to Alanine was less stable. The binding affinity of this mutant Rv2615c
with CARD-APAF1 and with CARD-Caspase9 was also decreased. Mutant
Rv2615c protein bound to APAF1 CARD with energy score of 594.43 Kcal/mol
and with Caspase9 CARD with energy score of -646.87 Kcal/mol.