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Structure of the apoptotic protease-activating factor 1 bound to ADP


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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Figure 1: Overall structure of the WD40-deleted Apaf-1 bound to ADP.
Figure 2: The CARD domain of Apaf-1 exists in a closed conformation.
Figure 3: ADP serves as an organizing centre for the adjoining three domains and locks Apaf-1 in an inactive conformation.
Figure 4: Apaf-1 is an active ATPase, and its ATPase activity seems to be essential to the activation of caspase-9.
Figure 5: A model for the formation of the apoptosome.

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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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Correspondence to Yigong Shi.

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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).

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