The importance of the dimer interface in the folding and assembly of procaspase-3.
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Date
2009-11-16
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Abstract
Caspases are a family of cysteine proteases that are intimately involved in apoptosis and exist in the cell as inactive zymogens prior to activation. Initiator procaspases are monomers that must dimerize for activation. Executioner procaspases, such as procaspase-3, are dimers that must be processed for activation. The chemical properties of the dimer interface are different between the two subfamilies of caspases but their structures are similar, suggesting that the interface region is important for regulation. The goal of the studies presented here is to determine the importance of the dimer interface in folding and assembly of procaspase-3. A histidine mutation was introduced into the dimer interface region, which completely abolished the activity of mature caspase-3. Equilibrium and kinetic folding studies were performed to elucidate how a mutation in the dimer interface prevents substrate turnover in the active site when the distance between the two regions is 20Ã…. The folding studies presented here coupled with the crystal structure show that the protein is entering a kinetic trap prior to dimerization because the histidine has to adopt an unusual rotomer to pack into a region that normally accommodates a much smaller valine residue. A hysteresis was observed by equilibrium folding studies, presumably because the time it takes to refold is different that the time it takes to unfold due to the limited conformational freedom of the histidine residues. Additionally, the hysteresis is observed to be concentration dependent suggesting that the histidine residue makes the activation mechanism of procaspase-3 more like initiator caspases that require a large local concentration of protein to promote dimerization. Kinetic refolding studies showed that the procaspase-3 monomer is becoming trapped in a conformation that is unstable and prone to aggregation prior to forming a dimerization competent species. The crystal structure of caspase-3 (V266H) revealed two separate pathways of inhibition starting from the dimer interface and culminating in the active site that could be responsible for the lack of activity in this mutant. These data, taken together, suggest that the dimer interface is a region that can be used to allosterically inhibit procaspase-3 because it is important for regulation of the enzyme. This is important because it could be used as a drug target for diseases that have too much cell death, such as neurodegenerative disorders.
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Keywords
dimerization, hysteresis, kinetic folding, caspase, apoptosis, equilibrium folding, protein folding
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Degree
PhD
Discipline
Biochemistry
