A novel BH3-like domain in BID is required for intramolecular interaction and autoinhibition of pro-apoptotic activity.

Upon activation of the Fas apoptotic signaling pathway, Bid, a "BH3 domain-only" pro-apoptotic molecule, is cleaved by caspase-8 into a 6.5-kDa N-terminal and a 15-kDa BH3 domain-containing C-terminal fragment, referred to as t(n)-Bid and t(c)-Bid, respectively. t(c)-Bid is a more potent inducer of apoptosis than full-length Bid, suggesting that the N-terminal region of Bid has an inhibitory effect on its pro-apoptotic activity. Here, we report the identification of a novel BH3-like motif (amino acid residues 35-43) in t(n)-Bid. Although Bid does not homodimerize, t(n)-Bid is able to associate avidly with t(c)-Bid. Site-directed mutagenesis revealed that both the novel BH3-like and BH3 domains are necessary for direct binding between t(n)-Bid and t(c)-Bid. While full-length Bid does not associate with t(n)-Bid, substitution of Leu(35), a critical residue in mediating t(n)-Bid/t(c)-Bid interaction, with Ala in full-length Bid is sufficient to establish Bid/t(n)-Bid interaction. Interestingly, the L35A Bid mutant is as effective as t(c)-Bid in inducing apoptosis and binding Bcl-X(L). We propose that the intramolecular interaction involving the BH3-like and BH3 domains serves to regulate the pro-apoptotic potential of Bid.