TY - JOUR
T1 - Synthetic antibodies inhibit Bcl-2-associated X protein (BAX) through blockade of the N-terminal activation site
AU - Uchime, Onyinyechukwu
AU - Dai, Zhou
AU - Biris, Nikolaos
AU - Lee, David
AU - Sidhu, Sachdev S.
AU - Li, Sheng
AU - Lai, Jonathan R.
AU - Gavathiotis, Evripidis
N1 - Publisher Copyright:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2016/1/1
Y1 - 2016/1/1
N2 - 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, cytosolicBAXis activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore.HowtheBAXactivation 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 α1/α6 and the α1 -α2 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.
AB - 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, cytosolicBAXis activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore.HowtheBAXactivation 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 α1/α6 and the α1 -α2 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.
UR - http://www.scopus.com/inward/record.url?scp=84952891730&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84952891730&partnerID=8YFLogxK
U2 - 10.1074/jbc.M115.680918
DO - 10.1074/jbc.M115.680918
M3 - Article
C2 - 26565029
AN - SCOPUS:84952891730
SN - 0021-9258
VL - 291
SP - 89
EP - 102
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -