Abstract
Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli1. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria2. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-Å crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered α/β fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.
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Acknowledgements
We thank M. Becker, A. Saxena, K. Dedrick and L. Walsh for assistance, A. Wist for help with the TLC, L. Gu for help with the omit map, F. Hughson for critically reading the manuscript, and members of the Shi laboratory for discussion. This work was supported by the NIH. S.J.R. is a Fellow of the Leukemia and Lymphoma Society.
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Supplementary information
Supplementary Figure S1
Sequence alignment of the human Apaf-1 protein with its homologues in fish, fly and worm (CED-4). (JPG 492 kb)
Supplementary Figure S2
Apaf-1 can activate caspase-9 in a 1:1 complex. a, Apaf-1 is capable of binding to caspase-9 in the absence of ATP/dATP. (JPG 38 kb)
Supplementary Table S1
Diffraction data and refinement statistics. (DOC 22 kb)
Supplementary Figure Legends
Legends to accompany Supplementary Figures S1 and S2. (DOC 20 kb)
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Riedl, S., Li, W., Chao, Y. et al. Structure of the apoptotic protease-activating factor 1 bound to ADP. Nature 434, 926–933 (2005). https://doi.org/10.1038/nature03465
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DOI: https://doi.org/10.1038/nature03465
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