STRUCTURAL AND FUNCTIONAL COMPLEMENTATION OF AN INACTIVE BCL-2 MUTANTBY BAX TRUNCATION

Interactions among proteins in the Bcl-2 family regulate the onset of programmed cell death, Previous work has shown that the death-inhibiti ng family members Bcl-2 and Bcl-x(L) form heterodimers with the death- promoting homologue Bar and that certain site-directed mutants of Bcl- 2 and Bcl-x(L) lose both biological activity and the ability to bind B ax. To better understand the structural basis of heterodimer formation , we have used a yeast two-hybrid assay to screen for mutants of Bax t hat regain the ability to bind to these inactive Bcl-2(G145A) and Bcl- x(L)(G138A) mutants, This screen identified a series of C-terminally t runcated Fax molecules that contain complete BH3 (Bcl-2 homology domai n 3) domains but that have lost BH1 and BH2 sequences. These results i ndicate that while the Bcl-2 and Bcl-x(L) mutants fail to bind full-le ngth Bax, they still retain a binding site for the critical BH3 domain , This suggests that conformational constraints in full-length Pax reg ulate 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 Fax molecule, bu t does not block apoptosis induced by wild-type Bar. This demonstrates that cell protection can be effected by directly binding pro-apoptoti c members of the Bcl-2 family.