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

George Shapovalov; Abigaël Ritaine; Nadege Charlene Essonghe; Ian de Ridder; Hristina Ivanova; Spyridoula Karamanou; Anastassios Economou; Geert Bultynck; Roman Skryma; Natalia Prevarskaya

doi:10.37349/etat.2022.00088

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

Explor Target Antitumor Ther. 2022;3(3):375-391. doi: 10.37349/etat.2022.00088. Epub 2022 Jun 28.

Authors

George Shapovalov^#^{1

2}, Abigaël Ritaine^#^{1

2

3}, Nadege Charlene Essonghe^{1

2}, Ian de Ridder³, Hristina Ivanova³, Spyridoula Karamanou⁴, Anastassios Economou⁴, Geert Bultynck³, Roman Skryma^{1

2}, Natalia Prevarskaya^{1

2}

Affiliations

¹ Univ. Lille, Inserm, U1003 - PHYCEL - Physiologie Cellulaire, F-59000 Lille, France.
² Laboratory of Excellence, Ion Channels Science and Therapeutics, 59655 Villeneuve d'Ascq, France.
³ KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, B-3000 Leuven, Belgium.
⁴ KU Leuven, Department of Microbiology and Immunology, Rega Institute of Medical Research, Laboratory of Molecular Bacteriology, Herestraat 49, B-3000 Leuven, Belgium.

^# Contributed equally.

Abstract

Aim: Inositol 1,4,5-trisphosphate receptor (IP₃R) is a ubiquitous calcium (Ca²⁺) 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 IP₃R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IP₃R-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 IP₃R: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 IP₃R: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 IP₃R gating in the low open probability (P_o) regime, characteristic of the basal IP₃R 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 IP₃R activity and, thus of Ca²⁺ 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 IP₃R 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.

Keywords: Bcl-2; Cancer; IP3R; calcium; molecular dynamics; organelle membrane-derived patch-clamp.