However, the conclusions derived from these studies are at variance likely because of the complexity of the molecular mechanisms involved as well as the need to compare in vitro and in vivo data. Additional work is thus necessary to fully understand Bcl-2 interactions and their relation to programmed cell death. To gain insight into the structural and biophysical factors involved in Bcl-2 protein-protein binding, we report here the characterization of a novel interaction between the BH3-only protein Harakiri and the Bcl-2 member Diva. Harakiri localizes in membranes and exerts proapoptotic activity by interacting with survival Bcl-XL and Bcl-2. Harakiri has not been characterized at the structural level except for its C-terminal sequence, which is known from low-resolution techniques to adopt a-helical conformation in model membranes. Diva has also been found predominantly in membranes. However, little functional data on Diva is available. Specifically, previous independent reports indicate that Diva can have both pro- or antiapoptotic function. Diva has also been reported to bind antiapoptotic Bcl- XL, and the proapoptotic Bcl-2 members Bik and Bak, according to co-immunoprecipitation assays. In contrast, binding studies using isothermal titration MK-2206 2HCl calorimetry indicate that Diva does not bind peptides comprising the BH3 region of several proapoptotic Bcl-2 proteins, including Bak and Harakiri. On this basis it has been suggested that Diva is not functionally equivalent to other Bcl-2 proteins. However, the 3D structure of Diva is very similar to the known structures of other Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Our NMR data combined with the recently reported structure of Diva indicate that the interaction involves in Diva��s surface the same groove previously observed in all other known structures of antiapoptotic/BH3-peptide CX-4945 complexes, indicating that binding is specific. To illustrate the formation of the complex a 3D structural model of the heterodimer is built using molecular docking and the NMR data as restraints. Altogether, these results suggest that at the structural level Diva binds death-inducing Harakiri in a fashion similar to other antiapoptotic Bcl-2 proteins. In addition, structural studies on Harakiri were carried out using NMR and circular dichroism. The data show that Harakiri is largely unstructured with only a small population of residual a-helical conformation. This result indicates that Harakiri is an intrinsically disordered protein like several other members of the BH3-only subfamily.