Cucurbit[8]uril Reactivation of an Inactivated Caspase-8 Mutant Reveals Differentiated Enzymatic Substrate Processing

Abstract

Caspase-8 constructs featuring an N-terminal FGG sequence allow for selective twofold recognition by cucurbit[8]uril, which leads to an increase of the enzymatic activity in a cucurbit[8]uril dose-dependent manner. This supramolecular switching has enabled for the first time the study of the same caspase-8 in its two extreme states; as full monomer and as cucurbit[8]uril induced dimer. A mutated, fully monomeric caspase-8 (D384A), which is enzymatically inactive towards its natural substrate caspase-3, could be fully reactivated upon addition of cucurbit[8]uril. In its monomeric state caspase-8 (D384A) still processes a small synthetic substrate, but not the natural caspase-3 substrate, highlighting the close interplay between protein dimerization and active site rearrangement for substrate selectivity. The ability to switch the caspase-8 activity by a supramolecular system thus provides a flexible approach to studying the activity of a protein at different oligomerization states.

Keywords: caspases; cucurbit[8]uril; protein assembly; protein engineering; supramolecular chemistry.

Scheme 1

Library of engineered caspase‐8 (casp‐8) proteins and concept of casp‐8 activity regulation by monomer–dimer equilibrium and supramolecular rescue of dimerization and activity through twofold binding of cucurbit[8]uril to the N‐terminal FGG‐tags. Casp‐8(wt) is fully processed into the two subunits and the intrinsic monomer–dimer equilibrium is shifted to the active dimer upon cucurbit[8]uril binding; Casp‐8(F468A) is not proteolytically processed and cucurbit[8]uril binding does not induce a functional reorganization. Casp‐8(D384A) features only a single cleavage site resulting in the linker sequence remaining attached to the small subunit and inhibition of background dimerization and activity, which can be rescued by cucurbit[8]uril, leading to the formation of catalytically active dimers.

Figure 1

Dose‐dependent effect of cucurbit[8]uril on the enzymatic activity of FGGcasp‐8(wt) (0.15 μm, black) and FGGcasp‐8(D384A) (0.15 μm, light grey) for the Ac‐IETD‐AFC substrate. The error bars represent the standard deviation based on three measurements.

Figure 2

Cleavage activity of FGGcasp‐8 (wt) and FGGcasp‐8(D384A) (both at 0.15 μm) for casp‐3 (4 μm) in the: A, C) absence, and B, D) presence of cucurbit[8]uril (1 μm). Casp‐3 fl (full length); ls (large subunit); ss (small subunit).