期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 291, 期 1, 页码 89-102出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M115.680918
关键词
antibody engineering; B cell lymphoma 2 (Bcl-2) family; BAX; mitochondrial apoptosis; protein structure; structure-function; protein targeting; synthetic antibody; phage display
资金
- National Institutes of Health [R00HL095929, R01CA178394, R21CA155472, R01 GM020501, R56AI110750, R01AI101436, 1S10OD016305, P30 CA013330]
- New York Structural Biology Center (NYSBC)
- Empire State Development's Division of Science, Technology and Innovation (NYSTAR)
- National Institutes of Health MSTP Training Grant [T32GM007288]
- NCI/National Institutes of Health Individual Training Fellowship [F31CA186672]
- Sidney Kimmel Foundation for Cancer Research
- Gabrielle's Angel Foundation for Cancer Research
- Alexandrine and Alexander L. Sinsheimer Foundation
- NATIONAL CANCER INSTITUTE [F31CA186672, R21CA155472, R01CA178394, P30CA013330] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R00HL095929] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI101436, R56AI110750] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM020501] Funding Source: NIH RePORTER
- OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH [S10OD016305] Funding Source: NIH RePORTER
The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices 1/6 and the 1-2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.
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