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Structural and functional complementation of an inactive Bcl-2 mutant by Bax truncation
1IDUN Pharmaceuticals, Inc., La Jolla, California 92037, USA. sottilie@idun.com
Abstract:
Interactions among proteins in the Bcl-2 family regulate the onset of programmed cell death. Previous work has shown that the death-inhibiting family members Bcl-2 and Bcl-xL form heterodimers with the death-promoting homologue Bax and that certain site-directed mutants of Bcl-2 and Bcl-xL lose both biological activity and the ability to bind Bax. To better understand the structural basis of heterodimer formation, we have used a yeast two-hybrid assay to screen for mutants of Bax that regain the ability to bind to these inactive Bcl-2(G145A) and Bcl-xL(G138A) mutants. This screen identified a series of C-terminally truncated Bax molecules that contain complete BH3 (Bcl-2 homology domain 3) domains but that have lost BH1 and BH2 sequences. These results indicate that while the Bcl-2 and Bcl-xL mutants fail to bind full-length Bax, they still retain a binding site for the critical BH3 domain. This suggests that conformational constraints in full-length Bax regulate its ability to bind to other Bcl-2 family members. Furthermore, we demonstrate that the normally inert Bcl-2(G145A) mutant effectively blocks apoptosis induced by a C-terminally truncated Bax molecule, but does not block apoptosis induced by wild-type Bax. This demonstrates that cell protection can be effected by directly binding pro-apoptotic members of the Bcl-2 family.
Insights
Inactive Bcl-2 and Bcl-xL mutants bind Bax BH3 domains, revealing conformational constraints regulate protein interactions. This binding can block apoptosis, demonstrating a new mechanism for cell protection.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Proteins in the B-cell lymphoma 2 (Bcl-2) family control programmed cell death.
- Death-inhibiting proteins Bcl-2 and Bcl-xL bind to the death-promoting protein Bax.
- Mutations in Bcl-2 and Bcl-xL can abolish their biological activity and Bax binding.
Purpose of the Study:
- To investigate the structural basis of heterodimer formation between Bcl-2 family proteins.
- To identify Bax mutants that can bind to inactive Bcl-2 and Bcl-xL mutants.
- To understand how conformational changes in Bax affect its interactions with other Bcl-2 family members.
Main Methods:
- Yeast two-hybrid assay to screen for Bax mutants.
- Site-directed mutagenesis of Bcl-2 and Bcl-xL proteins.
- Analysis of protein-protein interactions and apoptosis induction.
Main Results:
- Inactive Bcl-2(G145A) and Bcl-xL(G138A) mutants bind to C-terminally truncated Bax molecules containing the BH3 domain.
- Full-length Bax mutants that bind to inactive Bcl-2/Bcl-xL were identified, indicating the BH3 domain is critical for binding.
- Inactive Bcl-2(G145A) blocked apoptosis induced by truncated Bax but not wild-type Bax.
Conclusions:
- Conformational constraints in full-length Bax regulate its binding to other Bcl-2 family members.
- The BH3 domain of Bax is essential for binding to inactive Bcl-2 and Bcl-xL.
- Direct binding to pro-apoptotic proteins can mediate cell protection, offering a novel therapeutic strategy.