Helix 6 of tBid is necessary but not sufficient for mitochondrial binding activity.

The apoptosis effector Bid regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 alpha-helices (designated H1 through H8, respectively) and is a soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid yields activated "tBid" (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. Here, we demonstrate that helix H6 of tBid is necessary, albeit not sufficient, for mitochondrial binding. In particular, a 33 amino acid long domain, which encompassed H6 and H7, behaved as the minimum domain in tBid that was sufficient for mitochondrial binding. Unexpectedly, the hydrophobic surface of these helices could be mutated without altering the binding activity of the domain, implying that the secondary structure of the helices may be the key determinant of binding. These experiments expand our mechanistic understanding of the apoptotic regulator, tBid.