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Updated: Aug 1, 2026
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
Extended substrate recognition in caspase-3 revealed by high resolution X-ray structure analysis
Rajkumar Ganesan1, Peer R E Mittl, Stjepan Jelakovic
1Biochemisches Institut, Universität Zürich, Switzerland.
Abstract:
Caspases are cysteine proteases involved in the signalling cascades of programmed cell death in which caspase-3 plays a central role, since it propagates death signals from intrinsic and extrinsic stimuli to downstream targets. The atomic resolution (1.06 Angstroms) crystal structure of the caspase-3 DEVD-cmk complex reveals the structural basis for substrate selectivity in the S4 pocket. A low-barrier hydrogen bond is observed between the side-chains of the P4 inhibitor aspartic acid and Asp179 of the N-terminal tail of the symmetry related p12 subunit. Site-directed mutagenesis of Asp179 confirmed the significance of this residue in substrate recognition. In the 1.06 Angstroms crystal structure, a radiation damage induced rearrangement of the inhibitor methylketone moiety was observed. The carbon atom that in a substrate would represent the scissile peptide bond carbonyl carbon clearly shows a tetrahedral coordination and resembles the postulated tetrahedral intermediate of the acylation reaction.
Insights
Caspase-3, a key enzyme in programmed cell death, has its substrate selectivity revealed by a high-resolution crystal structure. This structure highlights the role of Asp179 in recognizing substrates for apoptosis signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Caspases are cysteine proteases crucial for programmed cell death (apoptosis).
- Caspase-3 is a central executioner protease, propagating death signals.
- Understanding caspase-3 substrate selectivity is vital for drug development and disease research.
Purpose of the Study:
- To elucidate the structural basis of substrate selectivity in caspase-3.
- To determine the atomic resolution crystal structure of the caspase-3 DEVD-cmk complex.
Main Methods:
- X-ray crystallography at 1.06 Angstroms resolution.
- Site-directed mutagenesis to probe residue function.
- Analysis of inhibitor-enzyme interactions.
Main Results:
- The crystal structure revealed a key low-barrier hydrogen bond between the P4 aspartic acid and Asp179.
- Asp179 was confirmed to be significant for substrate recognition via mutagenesis.
- Radiation damage revealed a tetrahedral intermediate mimic of the acylation reaction.
Conclusions:
- The S4 pocket's structural features, particularly Asp179, dictate caspase-3 substrate selectivity.
- The observed intermediate provides insights into the caspase catalytic mechanism.
- This structural data can inform the design of specific caspase inhibitors.
