Phage Display and Structural Studies Reveal Plasticity in Substrate Specificity and Allosteric Regulation of Caspase‐3 from Zebrafish

Abstract

The regulation of caspase enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. Caspase‐3, an effector caspase, is the primary executioner in apoptosis, but at sub‐threshold levels required for apoptosis, the activity of caspase‐3 is used by cells for a variety of physiological reactions (referred to as adaptive responses), from remodeling the cytoplasm, neuron pruning, receptor endocytosis, macrophage function, to development of eye lens and inner ear. The roles of caspases in apoptosis are well‐known, but their roles in adaptive responses are less clear, particularly in regard to how cells set the threshold of caspase activity to limit apoptosis while ensuring sufficient activity for signaling and differentiation. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3 (zCP3) from a cDNA library and examined substrate specificity of the recombinant protein compared to human caspase‐3 (hCP3) by utilizing novel M13 bacteriophage substrate specificity libraries that incorporated either random amino acids at P5‐P1’ or Asp fixed at P1. The results show that zebrafish caspase‐3 has similar preferences for P3‐P1’ amino acids, but the P4 position also accommodates hydrophobic amino acids. We determined the structure of zCP3 to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that amino acid substitutions at two sites may result in plasticity of the P4 amino acid by increasing flexibility of one active site loop. In addition, an allosteric site on the surface of the protein more closely resembles a similar site in caspase‐6, suggesting differential regulation when compared to the hCP3. The data suggest that zCP3 may exhibit a broader substrate portfolio, and while the known phosphorylation sites of hCP3 are conserved in zCP3, changes in an allosteric site on the protein surface, together with changes in substrate specificity, suggest overlap with the functions of caspase‐6 in zebrafish development.