Allosteric modulation of caspase 3 through mutagenesis

Abstract

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

Effect of a Procaspase-Activating Compound on the Catalytic Activity of Mature Caspase-3

“…Consequently, the interaction of 1 with Glu124/Arg164 will affect the positioning of Cys163 at the active site. This result is similar to previously reported docking simulations for the complexes of synthetic molecules with mutant procaspase-3s, where the role of Arg164 in the rearrangement of the active site structure 5 and an allosteric effect 45 on the binding of synthetic molecules to the protomer interface were proposed. Considering the similarity between facts in this and previous reports, we propose that 1 can bind to the protomer interface of mature caspase-3 to bring about structural perturbation at the active site of mature caspase-3.…”

“…1,2 This enzyme is produced as a mature form through the activation of the corresponding zymogen (procaspase-3) with the help of other effector enzymes (caspase-9, granzyme B and so on). 3,4 The X-ray crystallographic structure of the mature caspase-3 displays a "homo-heterodimeric dimer": 5 A small domain (ca. 12 kDa) and large domain (ca.…”

Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

“…The central sheet appears to be flanked by helices on both sides (Figure 2A). Dali [23] and ProFunc [24] analyses identified caspases, particularly caspases 3, 7, 8 and 9, as the closest structural matches; for example, an RMSD of 1.96 Å over 167 residues was obtained for caspase 3 (PDB ID: 4EHK) [25]. The largest difference was observed at a long loop (Y73-T111) between the β2-strand and α2-helix.…”

Modifications to a common phosphorylation network provide individualized control in caspases

“…Molecular dynamics (MD) simulations were performed as described previously with GROMACS 2016, using the Amber99 force field and the TIP3P water model (61)(62)(63)(64)(65). All simulations started with the structure obtained from X-ray crystallography, as described above, and the inhibitor was removed from the structure file.…”

“…All simulations started with the structure obtained from X-ray crystallography, as described above, and the inhibitor was removed from the structure file. As described previously for human caspase-3, the proteins were solvated in a periodic box of 62 Å x 48 Å x 66 Å, with approximately 13,500 water molecules (65). Sodium or chloride ions were added as required to neutralize the charge on the system.…”