title: Characterization of a novel interaction between Bcl-2 members Diva and Harakiri.
abstract: Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Sborgi
authors.forename: Susana
authors.initials: S
authors.lastname: Barrera-Vilarmau
authors.forename: Patricia
authors.initials: P
authors.lastname: Obregón
authors.forename: Eva
authors.initials: E
authors.lastname: de Alba
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D016923:Cell Death; D002470:Cell Survival; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D051017:Apoptosis Regulatory Proteins; C117144:BCL2-like 10 protein; C484586:HRK protein, human; D019253:Proto-Oncogene Proteins c-bcl-2