Structural and functional complementation of an inactive Bcl-2 mutant by Bax truncation.
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.
Ottilie
S
S
IDUN Pharmaceuticals, Inc., La Jolla, California 92037, USA. sottilie@idun.com
Diaz
J L
JL
Chang
J
J
Wilson
G
G
Tuffo
K M
KM
Weeks
S
S
McConnell
M
M
Wang
Y
Y
Oltersdorf
T
T
Fritz
L C
LC
D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein