Mitochondrial complexes containing the pro-apoptotic Bcl-2-family protein Bim: structure and regulatory function

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Mitochondrial apoptosis is a common form of regulated cell death in the immune system and other situations of biology. Mitochondrial apoptosis is regulated through a complex network of pro- and anti-apoptotic proteins, mostly members of the Bcl-2-family. Within the Bcl-2-family, two pro-apoptotic groups (the BH3-only initiators and the Bax/Bak effectors) and one anti-apoptotic group (five ‘Bcl-2-like’ proteins) regulate the release of cytochrome c, a decisive step of apoptosis. Many details about Bcl-2-family proteins are known, but substantial aspects of their molecular interactions and activity are still unclear. This uncertainty is linked to the multitude of potential interactions within the Bcl-2-family, generating a network that is very difficult to control and to understand in vivo. We have observed that a main trigger of mitochondrial apoptosis, the BH3-only protein Bim, is found on the outer mitochondrial membrane not as monomers but in a large complex, coordinated by the small protein dynein light chain 1 (DLC1), and specifically binding the anti-apoptotic Bcl-2-like protein Mcl-1. A second BH3-only protein, Bmf, was also recruited into these complexes. We have re-built Bim-containing complexes in vitro, where we have found that Bim-interactions with both DLC1 and lipid membranes are required for complex assembly. We have obtained data on structural changes induced in Bim by the binding of DLC1 and have started a structural analysis of Bim-DLC1-complexes. The goal of this project is to arrive at a structural understanding of Bim and Bim-containing complexes and to advance our understanding of the activation of Bim during apoptosis, focussing on early lymphocytes. We will use NMR and the Bim-binding partners Bmf, DLC1 and Mcl-1 in vitro to characterize spatial structure that is induced into the intrinsically disordered Bim protein by their binding. We will continue purifying in vitro-reconstituted Bim-DLC1-complexes with the aim to obtain high-resolution structure by cryo-EM. These studies will inform mutation approaches to investigate the functional relevance of identified structural and interaction sites. In situations of Bim-dependent apoptosis in lymphocytes we will then study the activation of Bim by small-molecule Bcl-2-antagonists or by the initiation of Bim-dependent apoptosis through upstream pro-apoptotic stimuli, observing changes in the composition of the complexes. In these experiments we will use an experimental system of mouse lymphocyte progenitor cells and early B cells. All established models of mitochondrial apoptosis assume one-on-one interactions between Bcl-2-family members to initiate or to block apoptosis. Our data indicate that signal transduction in the Bcl-2-family is even more complex, and we believe that the identified Bim-complexes are an important feature in the regulation of mitochondrial apoptosis. We hope that the experiments proposed here will clarify some of the currently unresolved issues.