Allosteric sensitization of proapoptotic BAX


BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3–α4 and α5–α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1–α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.

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Figure 1: STD NMR–based identification of BAX-interacting fragments that modulate BH3-mediated BAX activation.
Figure 2: Validation of BIF-44 as a dose-responsive binder and sensitizer of BAX.
Figure 3: BIF-44 targets the vMIA-binding region of BAX.
Figure 4: Allosteric deprotection of the α1–α2 loop and BAX BH3 domain upon BIF-44 binding.
Figure 5: BIF-44 sensitizes the BH3-triggered conformational activation and cytochrome c release activity of BAX.

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We thank E. Smith for graphics support; C. Sheahan for operational assistance with the NMR screen; M. Godes for assistance with mitochondrial preparation; G. Bird and T. Oo for peptide production; M. Ericsson and Z. Hauseman for technical assistance with electron microscopy; H.-S. Seo and S. Dhe-Paganon for ITC experiments; and D. Andrews (Sunnybrook Research Institute) and K. Sarosiek (Harvard T.H. Chan School of Public Health) for providing BIML plasmid and protein, respectively. This research was supported by NIH grant 1R35CA197583, a Leukemia and Lymphoma Society (LLS) Scholar Award, the Todd J. Schwartz Memorial Fund, the Wolpoff Family Foundation, and a grant from the William Lawrence and Blanche Hughes Foundation to L.D.W.; NIH grant R01GM101135 to J.R.E. and a research collaboration with the Waters Corporation (J.R.E.); NIH grant F31CA189651 to J.R.P.; an Alexander von Humboldt Foundation Feodor Lynen Fellowship to F.W.; and NIH training grant T32GM007753 to J.L.

Author information




J.R.P., F.W., J.L., G.J.H., W.M., S.L., T.E.W., J.R.E., and L.D.W. designed the study; J.R.P., F.W., and D.T.C. generated BAX protein, and J.R.P. conducted NMR experiments under the guidance of G.J.H., W.M., J.L., and L.D.W.; G.J.H., P.C., and W.M. analyzed screening results using software developed by P.C.; J.R.P. and F.W. performed biochemical and mitochondrial assays; J.L. performed the docking calculations and molecular dynamics simulations; S.L. and T.E.W. executed the HXMS experiments under the guidance of J.R.E.; all authors analyzed the data; and J.R.P., F.W., and L.D.W. wrote the manuscript, which was reviewed by all co-authors.

Corresponding author

Correspondence to Loren D Walensky.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–12 (PDF 15465 kb)

Molecular dynamics simulation of BAX (PDB ID 1F16) in solution (100 ns)

The D1–D2 loop (yellow; top of screen) shows relatively little motion and remains fixed over the D1/D6 (orange/cyan) trigger site at the N-terminal face of BAX ("closed" conformation). (MOV 7329 kb)

Molecular dynamics simulation of BIF-44-bound BAX in solution (100 ns)

Compared to unliganded BAX, there is increased flexibility and intermittent release of the D1–D2 loop (yellow; top of screen) from the D1/D6 (orange/cyan) trigger site, where BH3 interaction and displacement of the loop to the "open" conformation represents the initiating structural change of direct BAX activation. BIF-44 (stick representation) remains stablypositioned at an allosteric binding site formed by the junction of the D3/D4 and D5/D6 hairpins, distant from the D1–D2 loop. (MOV 6394 kb)

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Pritz, J., Wachter, F., Lee, S. et al. Allosteric sensitization of proapoptotic BAX. Nat Chem Biol 13, 961–967 (2017).

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