A Novel BH3-like Domain in BID Is Required for Intramolecular Interaction and Autoinhibition of Pro-apoptotic Activity

Abstract 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 tn-Bid and tc-Bid, respectively. tc-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 tn-Bid. Although Bid does not homodimerize, tn-Bid is able to associate avidly with tc-Bid. Site-directed mutagenesis revealed that both the novel BH3-like and BH3 domains are necessary for direct binding between tn-Bid and tc-Bid. While full-length Bid does not associate with tn-Bid, substitution of Leu35, a critical residue in mediating tn-Bid/tc-Bid interaction, with Ala in full-length Bid is sufficient to establish Bid/tn-Bid interaction. Interestingly, the L35A Bid mutant is as effective as tc-Bid in inducing apoptosis and binding Bcl-XL. We propose that the intramolecular interaction involving the BH3-like and BH3 domains serves to regulate the pro-apoptotic potential of Bid.