2.4 Rv2615c has CARD-like domain similar to CARD domains in
APAF1 and Caspase9
In-silico clues pointed the similarity of Rv2615c protein with
apoptosome complex and prediction of molecular function in activation of
cysteine-type endopeptidase/Caspases during apoptosis. In-vitro studies
substantiated the role of Rv2615c protein in inducing Caspase-mediated
host macrophage apoptosis. APAF1-Apoptosome complex initiates the
activation of Caspase9 and active Caspase9 is recruited via its CARD
domain onto the CARD domain of APAF1. Our findings of Rv2615c led us to
hypothesize- whether Rv2615c possess a CARD-like domain similar to CARD
domains present in APAF1 and Caspase9 that facilitates interaction and
activation of cysteine type endo-peptidase activity of Caspase9.
Multiple sequence alignment of Rv2615c protein with CARD domains of
APAF1 and Caspase9 revealed many conserved and highly similar amino acid
residues (Fig 5b and 5c). CARD domains of both APAF1 and Caspase9 are
known to contain some highly conserved mostly hydrophobic and crucial
residues through which protein-protein interactions occur (Fig 5a). We
observed 14 crucial residues of APAF1-CARD and 9 crucial residues of
Caspase9-CARD either totally conserved or highly similar in Rv2615c
protein positioned from 60 to 230 amino acids which we denoted as
CARD-like domain (Fig 5b, 5c and S4a). Interestingly, the MINNOU server
predicted that the CARD-like domain in Rv2615c is mostly hydrophobic in
nature similar to the CARD domains of APAF1 and Caspase9 (Fig S4b). The
secondary structure characterization reveals that the CARD-like domain
in Rv2615c contains highly disordered random coiled region and fewer
alpha-helixes (Fig S4b). Structural superimposition showed helical
region of CARD-like domain in Rv2615c mostly aligned with CARD-APAF1 and
CARD-Caspase9 (Fig S5). Coiled regions are highly flexible and unstable
which facilitate dynamic protein-protein interactions.