BAX activation is initiated at a novel interaction site

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Abstract

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized α-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB–BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

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Figure 1: NMR analysis of BAX on BIM SAHB titration.
Figure 2: Orientation of BIM SAHB at the BAX binding site.
Figure 3: BIM SAHB directly initiates BAX activation in vitro.
Figure 4: Sequence specificity of BIM-SAHB-induced BAX activation.
Figure 5: Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosis.

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Protein Data Bank

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The structural coordinates have been submitted to the Protein Data Bank under accession number 2K7W.

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Acknowledgements

We thank E. Smith for editorial and graphics assistance, W. Beavers for amino acid analyses, A. Perry for technical assistance, C. Turner and A. Bielecki of the MIT/Harvard Center for Magnetic Resonance for NMR technical advice, and R. Youle for feedback on the manuscript. We acknowledge the indelible contributions of the late S. J. Korsmeyer, who inspired this work. L.D.W. is supported by National Cancer Institute (NCI) grant 5P01CA92625, a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, a Culpeper Scholarship in Medical Science from the Goldman Philanthropic Partnerships, an American Society of Hematology Junior Faculty Scholar Award, and a grant from the William Lawrence Children’s Foundation. This research was also supported by NCI grant 5RO1CA50239. N.T. is supported by the Intramural Research Program of the National Heart, Lung and Blood Institute, NIH. E.H.-Y.C. is supported by the Searle Scholars Program and NCI grant 5RO1CA125562.

Author Contributions G.H.B. and L.D.W. designed, synthesized and characterized the SAHBs for structural and biological studies. M.S. and N.T. performed the NMR analysis of BAX and BIM SAHB, and E.G. and L.D.W. conducted the PRE NMR analysis of BAX using MTSL-derivatized SAHBs and performed the structure calculations. E.G., M.L.D. and K.P. executed the in vitro BAX activation studies. H.-C.T., H.K. and E.H.-Y.C. generated the BAX-reconstituted DKO MEFs and analysed their response to staurosporine, and S.G.K. examined the cellular response to BIM SAHB treatment.

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Correspondence to Nico Tjandra or Loren D. Walensky.

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L.D.W. is a scientific advisory board member and consultant for Aileron Therapeutics, Inc.

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Gavathiotis, E., Suzuki, M., Davis, M. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076–1081 (2008) doi:10.1038/nature07396

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