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Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Insights
Apoptosis relies on Apaf-1 binding caspase-9 via their Caspase Recruitment Domains (CARDs). This study reveals the Apaf-1 CARD structure and interaction surface, highlighting key hydrophobic and acidic residues crucial for this interaction.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Apoptosis regulation is crucial for cellular homeostasis and disease.
- Caspase activation, particularly caspase-9, is a key event in the intrinsic apoptosis pathway.
- The interaction between Apaf-1 CARD and caspase-9 CARD mediates caspase activation following mitochondrial damage.
Purpose of the Study:
- To determine the solution structure of the Apaf-1 CARD domain.
- To map the interaction surface between Apaf-1 CARD and caspase-9 CARD.
- To elucidate the molecular mechanisms driving the homophilic CARD/CARD interaction.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy
- Chemical shift perturbation mapping
- Structural modeling
Main Results:
- The Apaf-1 CARD domain comprises six alpha-helices with a unique kink in the N-terminal helix.
- The interaction interface involves an acidic surface patch on Apaf-1 CARD, centered on helices 2 and 3.
- Both electrostatic and hydrophobic interactions contribute significantly to the Apaf-1 CARD/caspase-9 CARD binding.
Conclusions:
- The structural and interaction data provide critical insights into the Apaf-1-mediated activation of caspase-9.
- Understanding the CARD/CARD interaction mechanism is vital for dissecting apoptosis signaling.
- This study proposes a model for the homophilic CARD/CARD interaction, integrating electrostatic and hydrophobic forces.
Abstract:
Direct recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD/CARD (Caspase Recruitment Domain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic alpha-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD/CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD/CARD interaction between Apaf-1 and caspase-9.