Modifications to a common phosphorylation network provide individualized control in caspases

Abstract

Caspase-3 activation and function has been well defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation.Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, S150 evolved with the apoptotic caspases, while T152 is a more recent evolutionary event in mammalian caspase-3. Substitutions at S150 result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of T152 in the conserved loop introduces a "kill switch" in mammalian caspase-3 while the more ancient S150 reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in both a common pathway for lowering activity during development as well as introducing a more recent cluster-specific switch to abolish activity.

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

“…Amino acid positions that were ambiguous were mostly found in the surface helices as well as two regions that are flexible or disordered, the intersubunit linker and prodomain (type A2 ambiguity). The surface helices were previously characterized as sites of allosteric regulation [22,60,61], so our data suggest that allosteric sites may have different evolutionary pressures compared to the active site residues. That is, the allosteric sites may have evolved in a species-dependent manner based on individual needs, leading to larger sequence variations at those sites and higher ambiguity in the APR analysis.…”

“…Caspases cleave target proteins through recognition of a tetrapeptide motif with the noted exception of caspase-2, which recognizes a pentapeptide sequence [20]. In some cases, enzyme specificity is coupled to exosites that facilitate substrate selection [21][22][23][24][25].…”

Remodeling hydrogen bond interactions results in relaxed specificity of Caspase-3

“…That is, the core of the proteins as well as the active sites are reasonably wellconserved compared to the surface helices, which show much lower conservation. As we and others showed previously, the helices are part of allosteric networks which may be fine-tuned for species-specific function (14)(15)(16). Thus, the lower conservation in the helices may reflect increased evolutionary pressure on allosteric regulation of the enzyme, compared to the more conserved active site.…”

Efficient synthesis of meso‐substituted porphyrins and molecular docking as potential new antioxidant and cytotoxicity agents

“…The bonds in the porphyrin ring were also rendered rigid during docking. We used the crystal structure 4MAN and 6BFJ for Bcl‐2 and caspase‐3, respectively. Docking was performed using 100 steps of genetic algorithm while keeping all the default settings provided by AutoDock Tools.…”