Allosteric cross-talk between the hydrophobic cleft and the BH4 domain of Bcl-2 in control of inositol 1,4,5-trisphosphate receptor activity.

AIM

Inositol 1,4,5-trisphosphate receptor (IPR) is a ubiquitous calcium (Ca) channel involved in the regulation of cellular fate and motility. Its modulation by anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) plays an important role in cancer progression. Disrupting this interaction could overcome apoptosis avoidance, one of the hallmarks of cancer, and is, thus, of great interest. Earlier reports have shown the involvement of both the Bcl-2 homology 4 (BH4) and the transmembrane domains (TMDs) of Bcl-2 in regulating IPR activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IPR-independent action at the mitochondria (Oncotarget. 2016;7:55704-20. doi: 10.18632/oncotarget.11005). The aim of this study was to investigate how targeting the BH3 hydrophobic cleft of Bcl-2 affects IPR:Bcl-2 interaction.

METHODS

Organelle membrane-derived (OMD) patch-clamp and circular dichroism (CD) thermal melting experiments were used to elucidate the effects of the ABT-199 (venetoclax) on the IPR:Bcl-2 interaction. Molecular dynamics (MD) simulations of free and ABT-199 bound Bcl-2 were used to propose a molecular model of such interaction.

RESULTS

It was shown that occlusion of Bcl-2's hydrophobic cleft by the drug ABT-199 finely modulates IPR gating in the low open probability (P) regime, characteristic of the basal IPR activity in non-excited cells. Complementary MD simulations allowed to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.

CONCLUSIONS

Bcl-2 is an important regulator of IPR activity and, thus of Ca release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Thus, it was found that the occlusion of the hydrophobic cleft of Bcl-2 by ABT-199 disrupts IPR activity, leading to Bcl-2 rebinding with smaller affinity and lesser inhibitory effect. MDs simulations of free and ABT-199 bound Bcl-2 propose a molecular model of such disruption, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.