A Tunable Allosteric Library of Caspase‐3 Identifies Coupling Between Conserved Water Molecules and Conformational Selection

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformer, while targeting an allosteric site in the dimer interface selects an inactive conformer that contains disordered active site loops. Mutations and post‐translational modifications stabilize high‐energy inactive conformers, with mostly formed, but distorted, active sites. In order to examine the interconversion of active and inactive states in the ensemble, we used DRoP to analyze 4,995 waters in 15 high‐resolution (<2.0 Å) structures of wild‐type caspase‐3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of post‐translational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of nineteen caspase‐3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies over four orders of magnitude. Altogether, our database consists of 37 high‐resolution structures of caspase‐3 variants, and we demonstrate that each 10‐fold decrease in activity correlates with a loss of 23 conserved water molecules. The data show that the activity of caspase‐3 can be fine‐tuned through globally de‐solvating the active conformer within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine‐tune activity through conformational selection.