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

Apoptotic caspases exist not as static structures but as dynamic ensembles in solution, finely tuned by post-translational modifications and oligomerization. The fine-tuning of this ensemble by cellular cues allows caspases to influence not only apoptotic pathways but also the non-apoptotic pathways in which they are involved. These ensembles span a complex conformational landscape from well-characterized low-energy states captured in structural databases to transient high-energy intermediates that remain elusive and poorly understood. This limited structural view poses a major barrier to fully understanding how caspase activity is regulated and diversified across cellular contexts. To address this, we integrate evolutionary, folding, and mutational data with molecular dynamics simulations and network analysis to uncover a highly conserved residue network in structural space that has been faithfully passed on in sequence space over 500 million years of vertebrate evolution. This network encodes a high-energy intermediate consistently present in the ensemble of all present-day vertebrate apoptotic caspases. It not only guides folding but also scaffolds dynamic motions, functioning like a structural backbone that supports the ensemble. Building on this foundation, we identify differentially evolving networks surrounding the conserved core in initiator and effector caspase subfamilies. These variations provide thermodynamic insight into how initiators stabilize monomeric conformations while effectors favor dimeric states, revealing how evolution shapes ensembles to diversify function in protein families. Additionally, we discover conserved hub residues near an allosteric hotspot, distinct from the core network, that regulate the dynamics of surrounding evolving networks and act as control centers that modulate the conformational equilibrium within the apoptotic caspase ensemble.