Molecular determinants of Bcl–2 and Bcl–Xl binding to the IP3R channel and regulation of its activity at the molecular and functional level in normal and apoptotic cells

B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra-large (Bcl-Xl) are major anti-apoptotic proteins, members of the Bcl-2 family, which have a key role in the regulation of cell-fate decisions. Bcl-2 and Bcl-Xl share all four B-cell homology (BH1-4) domains, with an N-terminal BH4 domain and a hydrophobic cleft formed by the BH3-BH2-BH1 domains. Bcl-2 and Bcl-Xl also possess a C-terminal trans-membrane domain (TMD), which is responsible for their targeting at different intracellular membranes like mitochondrial, endoplasmic reticulum (ER) or even nuclear membrane. In addition to opposing the pro-apoptotic members, thereby preventing their activation and subsequent cell death, Bcl-2 and Bcl-Xl execute their anti-apoptotic functions by regulating Ca2+ signals. Bcl-2 and Bcl-Xl emerged as direct functional regulators of the IP3 receptor (IP3R), the main intracellular Ca2+-release channel. Bcl-2 appears to function as an inhibitor of IP3R channel, suppressing pro-apoptotic Ca2+-release events mediated by IP3R. Depending on its concentration, Bcl-Xl can function as a sensitizer or inhibitor of IP3R channel. At low concentrations, Bcl-Xl sensitizes IP3R activity, which results in boosted mitochondrial metabolism and bio-energetics, while when present at high concentrations, Bcl-Xl inhibits IP3R activity, suppressing Ca2+-mediated apoptosis.

An increasing body of data suggests that the complex modulation of IP3Rs by Bcl-2 and Bcl-Xl proteins relies on multi-domain interactions. To date, 2 different IP3R regions have been identified as target sites for Bcl-2 and Bcl-Xl. Via its BH4 domain, Bcl-2 binds to the Domain 3 of IP3R, which is located in the central, modulatory domain. This interaction is considered as the driving force for the inhibition of IP3R activity by Bcl-2. Bcl-2 was also proposed to target the C-terminus of IP3R, but this remained poorly understood. Bcl-Xl was shown to interact with the same domains like Bcl-2, Domain 3 and C-terminus. Recently, it was reported that Bcl-Xl engages its hydrophobic cleft and to target the C-terminus of IP3R, but the binding determinants in Bcl-Xl/Domain 3 complex are still under investigation.

Previously, we showed that while Bcl-2 targets with similar efficiency the Domain 3 and the C-terminus of IP3R, Bcl-Xl shows preferences for the C-terminus. Here, we demonstrated that in contrast to Bcl-Xl, the hydrophobic cleft of Bcl-2 does not contribute to the binding to and regulation of IP3R. We revealed an interaction between the Bcl-2’s TMD and the C-terminus of IP3R, which was critical for an efficient IP3R inhibition and protection against Ca2+-mediated apoptosis by Bcl-2. Furthermore, we identified the ligand-binding domain of IP3R as a novel interaction partner of Bcl-2 and in particular, of its BH4 domain. We observed a mutual antagonism between Bcl-2/BH4 domain and the ligand, IP3 which were able to disrupt each other’s interaction with the ligand-binding domain. We anticipate that such an interaction might serve as a parallel or additional mechanism of IP3R inhibition by Bcl-2, thereby revealing an additional layer of regulation where the level of agonist stimulation modulates the inhibitory function of Bcl-2.

Finally, we confirmed the important role of the hydrophobic cleft of Bcl-Xl for IP3R regulation and identified another candidate for molecular determinant, involved in this complex. We showed that the Lys87, located in the BH3 domain, but outside of the hydrophobic cleft of Bcl-Xl, contributes to Bcl-Xl binding to IP3R and to the regulation of the channel.

In conclusion, we present a model where the 2 very similar anti-apoptotic proteins, Bcl-2 and Bcl-Xl, target the same domains in IP3R, but with different efficiencies and engaging distinct binding determinants, thereby resulting in a differential regulation of the IP3R-mediated Ca2+ signals.