Date of Award

6-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Tudor Moldoveanu, Ph.D.

Committee

Taosheng Chen, Ph.D.; Richard Kriwacki, Ph.D.; Ramesh Narayanan, Ph.D.; Joseph Opferman, Ph.D.; Gerard Zambetti, Ph.D.

Abstract

Mitochondrial apoptosis is crucial for normal mammalian development and adult homeostasis being regulated by B-Cell Lymphoma-2 (BCL-2) proteins. Deregulation of BCL-2 proteins form the basis for myriad of human pathologies including cancer and autoimmune diseases. Therefore, BCL-2 proteins are considered key therapeutic targets. BCL-2 homologous antagonist/killer (BAK) is an effector of outer mitochondrial membrane (OMM) pemeabilization or mitochondrial poration. Despite progress in understanding BAK-mediated mitochondrial poration, the underlying multi-step mechanism is not clearly defined. Here, we provide mechanistic details on the early steps in this process related to BAK activation, including transient direct activation and autoactivation. We demonstrate for the first time the consequences of direct BAK activation by BH3 ligands for BAK destabilization, which is a requisite for mitochondrial poration. Our high-resolution crystal structures of engineered BID BH3 ligands bound to BAK have captured the rearrangements of a buried electrostatic network at the base of hydrophobic groove, which explains the role of helix α1 destabilization in direct BAK activation. Furthermore, we defined a novel mechanism of BAK autoactivation in trans whereby the exposed BH3 domain (helix α2) engages dormant BAK in a “BH3-in-groove” conformation to amplify BAK activation in mitochondrial poration. The crystal structure of the BAK BH3-BAK complex unveils the structural basis of in trans autoactivation. The structure provides evidence for destabilization of the buried electrostatic network of helix α1 similar to that observed during direct BAK activation. We comprehensively validated this mechanism by mutagenesis in the context of BAK BH3 peptides and full-length BAK protein. Additionally, we probed BAK activation with small molecules identified in a fragment-based screen as well by modifying existing BCL-xL inhibitors. Our results suggest that alterations in helix α1 rearrangements are common basis for both direct activation by BH3 ligands and helix α2 mediated autoactivation of BAK. It is possible that both activation steps are required for formation of activated BAK intermediate which may interact with outer mitochondrial membrane to form pores and subsequently initiate mitochondrial apoptosis. Targeting both direct activation and autoactivation by mimicking the conformational changes induced by BH3 peptides changes could serve as a potential strategy for treating number diseases caused by deregulation of BCL-2 family proteins. Overall, our studies redefine BAK activation mechanistically and provide strong preliminary data towards the design of small molecule BAK modulators for apoptosis research.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0003-2388-995X

DOI

10.21007/etd.cghs.2020.0508

2020-017-Singh-DOA.pdf (388 kB)
Declaration of Authorship

Available for download on Saturday, June 25, 2022

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