Unrevealing the structural mechanisms of Bcl-2 : from functional modulation to complex formation with Beclin 1 and Bag-1

Abstract

Apoptosis and autophagy are two fundamental cellular processes that regulate cell fate. Apoptosis is a programmed cell death mechanism essential for development, tissue homeostasis, and immune responses, whereas autophagy ensures cellular homeostasis by degrading and recycling damaged organelles and proteins. These pathways share common regulatory components, with B-cell lymphoma 2 (Bcl-2) playing a crucial role as an anti-apoptotic protein. Bcl-2 inhibits programmed cell death and regulates cell survival and death by interacting with various proteins, such as Beclin 1 and Bag- 1. The balance between these processes is critical for cellular integrity, and dysregulation is frequently associated with various pathologies, including cancer and neurodegenerative disorders. This study aims to elucidate the structural and functional dynamics of Bcl-2 interactions with Beclin 1 and Bag-1 to gain mechanistic insights into its dual regulatory role in apoptosis and autophagy. The first chapter of this study focuses on the newly developed production and purification strategy of the full-length, folded and functional apo-Bcl-2, highlighting its conformational dynamics, emphasizing its transition between closed and open states. Molecular dynamics (MD) simulations and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) analyses identified a crucial conformational shift mediated by salt bridge interactions involving residues R110-E136 and D111-R146. These interactions regulate the accessibility of Bcl-2's ligand-binding cavity, suggesting that its structural conformation plays a significant role in its dual function. Additionally, the R110A mutation was introduced to disrupt the salt bridge network, resulting in an "open" conformation that enhances ligand binding. The findings indicate that the flexibility of Bcl-2 influences its ability to function in both apoptosis and autophagy, providing a molecular framework for understanding its interactions with other regulatory proteins. The second section investigates the Bcl-2/Beclin 1 complex, which is central to macro- autophagy regulation. HDX-MS analysis of full-length Beclin 1 revealed its time- dependent dynamic structure. Structural and biochemical assays identified responsible residues of both Bcl-2 and Beclin 1 for Bcl-2/Beclin 1 complex. The utilization of full- length structures for both Bcl-2 and Beclin 1 allowed for a comprehensive analysis of all relevant residues. The literature reports that Beclin 1 binds to the ligand binding cavity of Bcl-2 through a conserved BH3 domain interaction modulated by Bcl-2's conformational states. Consistent with previous literature, the BH3 domain of Beclin 1 has been identified as the primary interaction site in the Bcl-2/Beclin 1 complex. Also, key residues within Bcl-2's ligand-binding groove critical for Beclin 1 sequestration were identified. Furthermore, time-dependent HDX-MS analyses demonstrated alternative Bcl-2 binding sites on Beclin 1, which was also proved by the in vitro complex formation assays, probably to regulate autophagy intiation. Considering the limitations on the targeting of Bcl-2 ligand binding cavity, these newly identified regions may constitute new therapeutic targets for small molecule or peptide inhibitors to modulate autophagy in cancer and neurodegenerative diseases. The final section focuses on the interaction between Bcl-2 and Bag-1, a co-chaperone protein that enhances Bcl-2's anti-apoptotic function. According to the literature, Bag- 1 stabilizes Bcl-2 and promotes its association with apoptotic regulators. Using a combination of biochemical assays and structural analyses, distinct interaction surfaces between Bag-1 and Bcl-2 were identified. Our data also specified the role of ATP on the interaction of Bag-1 and Bcl-2. Taken together, the biochemical and structural analyses conducted in this study contribute to our understanding of the impact of Bcl-2's conformational flexibility on cellular homeostasis. Additionally, the interactions of Bcl-2 with Beclin 1 and Bag-1 have been comprehensively examined at both structural and functional levels, shedding light on the key regulatory mechanisms of apoptosis and autophagy. The findings reveal critical interaction sites in the Bcl-2/Beclin 1 and Bcl-2/Bag-1 protein- protein interactions that could serve as therapeutic targets. Based on these data, the development of therapeutic molecules specifically targeting the protein-protein interactions among Bcl-2, Beclin 1, and Bag-1 is expected to contribute to the treatment of various diseases, including cancer and neurodegenerative disorders.