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Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1
C L Day1, C Dupont, M Lackmann
1Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
Insights
The Apaf-1 CARD structure reveals key binding sites for procaspase-9, crucial for initiating apoptosis. This finding clarifies the molecular mechanism of apoptosis activation.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is a fundamental biological process.
- Activation of the apoptosis pathway often involves caspase cascades.
- Procaspase-9 activation requires interaction with the Apaf-1 adaptor protein via their respective CARDs.
Purpose of the Study:
- To determine the three-dimensional structure of the Apaf-1 CARD.
- To identify specific residues within the Apaf-1 CARD essential for binding procaspase-9.
- To elucidate the molecular basis of Apaf-1/procaspase-9 complex formation.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy was employed to determine the structure of the Apaf-1 CARD.
- Site-directed mutagenesis was used to identify critical binding residues.
- Structural comparisons were made with related protein domains.
Main Results:
- The Apaf-1 CARD structure consists of seven alpha-helices, with six arranged antiparallelly.
- Specific residues located in helix 2 of the Apaf-1 CARD are central to the interaction with procaspase-9 CARD.
- The identified interaction surface differs from that of RAIDD CARD but aligns with FADD DED and crystallographic data for Apaf-1 CARD.
Conclusions:
- The structural and mutagenesis data provide a detailed molecular understanding of Apaf-1 CARD and its interaction with procaspase-9.
- This research clarifies a critical step in the initiation of the intrinsic apoptosis pathway.
- The findings have implications for understanding caspase activation and developing related therapeutics.
Abstract:
Activation of procaspase-9, a key component of the apoptosis mechanism, requires the interaction of its caspase recruitment domain (CARD) with the CARD in the adaptor protein Apaf-1. Using nuclear magnetic resonance spectroscopy and mutagenesis we have determined the structure of the CARD from Apaf-1 and the residues important for binding the CARD in procaspase-9. Apaf-1's CARD contains seven short alpha-helices with the core six helices arranged in an antiparallel manner. Residues in helix 2 have a central role in mediating interaction with procaspase-9 CARD. This interaction surface is distinct from that proposed based on the structure of the CARD from RAIDD, but is coincident with that of the structurally similar FADD death effector domain and the Apaf-1 CARD interface identified by crystallographic studies.