The Bcl-2 family proteins regulate a major pathway for apoptosis that involves intermolecular associations with each other and with a variety of other partners, leading to the release of cytotoxic molecules from the mitochondria (1
). The proteins share sequence homology across four evolutionarily conserved Bcl-2 homology (BH1–BH4) domains, of which the BH3 domain is highly conserved and essential for both the cell killing activity of the pro-apoptotic family members, and for mediating protein–protein interactions among the various Bcl-2 proteins. Several proteins also contain a 20-residue hydrophobic C-terminus, believed to be responsible for their association with intracellular membranes. The six human antiapoptotic members of the family possess all four BH domains, while the pro-apoptotic family members have more diverse sequences. The latter set is subdivided into multi-domain proteins such as Bax and Bak, containing BH1, BH2, and BH3 domains, and BH3-only proteins such as Bid and Bim, containing only the BH3 domain. These BH3-only proteins are activated by upstream death signals, which trigger their transcriptional induction or post-translational modification, providing a key link between extrinsic stress cues, transmitted by the death receptors, and the mitochondrial pathway to cell death (2
Bid is a cytosolic protein that is cleaved and activated by caspases, calpains, and lysosomal proteases, after engagement of the Fas or TNFR1 cell surface receptors, which link it to both apoptotic and necrotic cell death pathways in stroke, neuro-degeneration, hepatitis, and other ailments (3
). Cleavage by caspase-8 produces the 15 kDa C-terminal fragment, tBid, which can interact with and be sequestered by the antiapoptotic proteins Bcl-2 and Bcl-XL
), or interact with the multi-domain pro-apoptotic proteins Bax and Bak to promote mitochondrial apoptosis (12
). In addition, tBid can translocate from the cytosol to mitochondria to induce mitochondrial membrane remodeling, the release of cytotoxic molecules, and ultimately, cell death (10
). The BH3 domain of Bid is essential for binding to other antiapoptotic or pro-apoptotic Bcl-2 family members, however, it is not required for translocation to the mitochondria or for mitochondrial membrane remodeling, which occurs independently of other Bcl-2 proteins.
The functions of Bid, Bim, and Bax can also be regulated by Humanin (HN), a 24-residue endogenous peptide that protects neuronal cells from a variety of toxic insults (13
). Humanin was first identified as an antiapoptotic factor capable of rescuing neuronal cells from apoptosis in familial Alzheimer's disease (13
), and has been reported to bind receptors in the plasma membrane (18
), as well as to regulate the pro-apoptotic activities of Bid, Bim, and Bax in the cytosol (20
). Although the structures of Bid, Bim, Bax, and Humanin have been determined or characterized independently, no molecular information has been available about the interaction of Humanin with its Bcl-2 partners, and its mechanism of Bcl-2-dependent cytoprotection has been unknown.
The structures of Bid (23
) and Bax (25
) are both strikingly similar to the structures of other multi-domain pro-apoptotic and antiapoptotic Bcl-2 family proteins, despite the low level of amino acid sequence homology, while Bim is largely unfolded (26
). Bid folds into eight α-helices, with the third helix coinciding almost entirely with the conserved BH3 domain. Humanin is unfolded in water but NMR shows that it adopts a helical structure in the presence of 30% trifluoroethanol, indicating a propensity for this type of secondary structure (27
), while at high salt concentrations CD analysis shows some evidence of extended conformation (29
In this report, we characterize the Humanin-binding site of Bid using NMR chemical shift mapping experiments. By monitoring the changes in the 1H / 15N heteronuclear single quantum correlation (HSQC) spectrum of Bid upon titration with peptides derived from Humanin we show that the interaction with Humanin affects Bid residues in the BH3 domain. The changes in 1H and 15N chemical shifts for all the titratable backbone amide signals can be modeled with a single binding equilibrium constant, demonstrating that Humanin binds specifically to Bid. The demonstration of a Humanin-specific binding site on the surface of Bid reinforces the role of this peptide as a direct modulator of Bid-dependent programmed cell death, and suggests a strategy for the development of therapeutic approaches for the many related diseases.