Modifying caspase-3 activity by altering allosteric networks.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Caspases possess allosteric sites that bind small molecules or peptides, influencing enzyme activity.
• Allosteric regulation typically involves conformational changes leading to disordered substrate-binding pockets.
• Mutations in caspase allosteric networks can reduce activity without significant structural alterations.

Purpose of the Study:

• Investigate the role of the allosteric network and helix 3 in caspase-3 activity.
• Determine the mechanism of inactivation by the V266H mutation in caspase-3.
• Characterize the conformational states of caspase-3.

Main Methods:

• Site-directed mutagenesis of caspase-3, focusing on the V266 residue and surrounding allosteric network.
• Enzyme activity assays to assess the impact of mutations.
• Structural analysis to evaluate conformational changes.

Main Results:

• The V266H mutation in caspase-3 introduces steric clashes, inactivating the enzyme despite an intact substrate-binding pocket.
• Restoring interactions by mutating residues near H266 in caspase-3 V266H reactivated the enzyme.
• Mimicking steric clashes elsewhere in the allosteric network also reduced caspase-3 activity.

Conclusions:

• Caspase-3 exists in an ensemble of conformations, including active and inactive states with intact substrate-binding pockets but altered helix 3.
• Enzyme activity is determined by the relative populations of these conformational states.
• Allosteric regulation of caspase-3 activity is mediated by structural elements distinct from the active site.