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Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1

Abstract

Caspase-9-mediated apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multicellular organisms. Mature caspase-9 is derived from its procaspase precursor as a result of recruitment by the activating factor Apaf-1. The crystal structures of the caspase-recruitment domain of Apaf-1 by itself and in complex with the prodomain of procaspase-9 have been determined at 1.6 and 2.5 Å resolution, respectively. These structures and other evidence reveal that each molecule of Apaf-1 interacts with a molecule of procaspase-9 through two highly charged and complementary surfaces formed by non-conserved residues; these surfaces determine recognition specificity through networks of intermolecular hydrogen bonds and van der Waals interactions. Mutation of the important interface residues in procaspase-9 or Apaf-1 prevents or reduces activation of procaspase-9 in a cell-free system. Wild-type, but not mutant, prodomains of caspase-9 completely inhibit catalytic processing of procaspase-9. Furthermore, analysis of homologues from Caenorhabditis elegans indicates that recruitment of CED-3 by CED-4 is probably mediated by the same set of conserved structural motifs, with a corresponding change in the specificity-determining residues.

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Figure 1: Structure of Apaf-1 CARD domain.
Figure 2: Representation of the structure of Apaf-1 CARD in complex with the procaspase-9 prodomain.
Figure 3: Sequence and structure conservation between Apaf-1 CARD and procaspase-9 prodomain.
Figure 4: Electrostatic interaction at the heterodimer interface.
Figure 5: Recognition specificity at the heterodimer interface.
Figure 6: Effect of point mutations on the catalytic activation of procaspase-9.
Figure 7: Predicted recognition interface between CED-3 and CED-4.

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Acknowledgements

We thank S. Kyin for peptide sequencing and mass spectroscopic analysis; F.Hughson for critically reading the manuscript; A. Saleh for purifying recombinant Apaf-1; R.Mukattash for technical assistance; and E. Steckman for secretarial assistance. This work was supported by start-up funds from Princeton University (to Y.S.) and grants from the NIH (to E.S.A.). Y.S. is a Searle Scholar and a Rita Allen Scholar.

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Correspondence to Yigong Shi.

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Qin, H., Srinivasula, S., Wu, G. et al. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature 399, 549–557 (1999). https://doi.org/10.1038/21124

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