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.