Sequential Unfolding Mechanisms of Monomeric Caspases.

Area of Science:

• Biochemistry
• Evolutionary Biology
• Molecular Biology

Background:

• Caspases are essential proteases regulating cell development and apoptosis.
• Apoptotic caspases exist as monomeric (initiator) or dimeric (effector) subfamilies, with distinct activation mechanisms.
• The evolutionary divergence of caspase subfamilies from a common ancestor is not fully understood.

Purpose of the Study:

• To investigate the folding landscape and stability of monomeric caspases from coral species.
• To understand the evolutionary relationship between monomeric and dimeric caspase subfamilies.
• To explore the pH-dependent conformational changes and regulation of monomeric caspases.

Main Methods:

• Examined protein folding stability across a wide pH range (3-10.5).
• Utilized molecular dynamics simulations, limited proteolysis, and MALDI-TOF mass spectrometry.
• Analyzed evolutionary divergence of coral and human caspases.

Main Results:

• Coral caspases exhibit high stability (∼15 kcal mol⁻¹) near physiological pH.
• Proteins unfold through two partially folded intermediates in equilibrium.
• A conserved allosteric site mediates pH-dependent conformational changes, with the small subunit unfolding before the large subunit.

Conclusions:

• All caspases share a conserved folding landscape.
• A conserved allosteric site allows for species-specific regulation.
• The evolution of stable dimeric caspases may be linked to stabilizing the small subunit.