Allosteric Tuning of Caspase-7: Establishing the Nexus of Structure and Catalytic Power.

Caspase-7 (C7), a cysteine protease involved in apoptosis, is a valuable drug target for its role in human diseases (e. g., Parkinson's, Alzheimer's, sepsis). The C7 allosteric site has great potential for small-molecule targeting, but numerous drug discovery efforts have identified precious few allosteric inhibitors. Here we present the first selective, drug-like inhibitor of C7 along with several other improved inhibitors based on our previous fragment hit. We also provide a rational basis for the impact of allosteric binding on the C7 catalytic cycle by using an integrated approach including X-ray crystallography, stopped-flow kinetics, and molecular dynamics simulations. Our findings suggest allosteric binding disrupts C7 pre-acylation by neutralization of the catalytic dyad, displacement of substrate from the oxyanion hole, and altered dynamics of substrate binding loops. This work advances drug targeting efforts and bolsters our understanding of allosteric structure-activity relationships (ASARs).

Determinants of human glucokinase activation and implications for small molecule allosteric control.

Glucokinase (GK) is an enzyme that catalyzes the ATP-dependent phosphorylation of glucose to form glucose-6-phosphate, and it is a tightly regulated checkpoint in glucose homeostasis. GK is known to undergo substantial conformational changes upon glucose binding. The monomeric enzyme possesses a highly exotic kinetic activity profile with an unusual sigmoidal dependence on glucose concentration. In this interdisciplinary study, which draws on small angle X-ray scattering (SAXS) integrated with 250 ns of atomistic molecular dynamics (MD) simulations and experimental glucose binding thermodynamics, we reveal that the critical regulation of this glucose sensor is due to a solvent controlled "switch". We demonstrate that the "solvent switch" is driven by specific protein structural dynamics, which leads to an enzyme structure that has a much more favorable solvation energy than most of the protein ensemble. These findings uncover the physical workings of an agile and flexible protein scaffold, which derives its long-range allosteric control through specific regions with favorable solvation energy. The physiological framework presented herein provides insights that have direct implications for the design of small molecule GK activators as anti-diabetes therapeutics as well as for understanding how proteins can be designed to have built-in regulatory functions via solvation energy dynamics.