Evolution of the conformational ensemble and allosteric networks of apoptotic caspases in chordates

Caspases, known for their role in apoptosis and inflammation, have recently emerged as potential regulators of cell differentiation and proliferation. The regulation of caspase conformations to affect these specific pathways is not well understood. Understanding the conformational landscapes, intermediate states, and mechanisms for fine-tuning these ensembles is critical for unraveling their diverse regulatory functions. We combined experimental and computational techniques to characterize the evolution of conformational ensemble in caspases. Using ancestral protein reconstruction, molecular dynamics simulations, and network analysis, we identify a network of residues that serve as a scaffold for conformational variation, and the network has been faithfully passed down over 500 million years of apoptotic caspase evolution. In addition, investigations of free energy landscapes provide thermodynamic insights into how initiator and effector caspase subfamilies stabilize monomeric and dimeric conformations while undergoing conformational dynamics. We find, through mutational in-vitro folding analysis, that the small subunit and amino acid networks at the base of the structure are essential for conformational dynamics beyond a ubiquitous intermediate present in the conformational ensemble of all apoptotic chordate caspases. We identify key allosteric hub residues that regulate the equilibrium in the conformational ensembles of all apoptotic caspases in chordates. Lastly, by combining our new data set with previous folding studies we provide a comprehensive model for folding of all apoptotic caspases in chordates.