Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax.
The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death. Expression of Bax in Saccharomyces cerevisiae as a membrane-bound fusion protein results in a lethal phenotype that is suppressible by co-expression of Bcl-2. Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bax-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196). In vitro binding experiments using GST-Bcl-2 fusion proteins demonstrated that Bcl-2(delta B) and Bcl-2(delta C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2. In contrast, Bcl-2(delta A) and an NH2-terminal deletion mutant Bcl-2(delta 1-82) retained Bax binding activity in vitro but failed to suppress Bax-mediated cytotoxicity in yeast. Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast. Analysis of Bcl-2/Bcl-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/Bcl-2 homodimerization and revealed that sequences within the NH2-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination. In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.
Hanada
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La Jolla Cancer Research Foundation, Oncogene and Tumor Suppressor Gene Program, California 92037, USA.
Aimé-Sempé
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C
Sato
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Reed
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JC
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques; D051028:bcl-2-Associated X Protein
D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein