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.

Design and recognition of cucurbituril-secured platinum-bound oligopeptides

“…This feature was discovered by Urbach and coworkers, [15][16][17][18][19][20][21][22][23] and was exploited by the same group to selectively encapsulate into CB [7] the N-terminal Phe residue of proteins, including the insulin B-chain, 24 the human growth hormone (hGH), 25 ubiquitin 26 and myoglobin. 26 Brunsveld and coworkers [27][28][29][30][31][32][33] showed that protein dimerization is possible by exploiting the recognition ability of CB [8] towards pairs of Phe residues, leading to applications such as enzyme activity modulation. 29,31 Liu also showed that the same motif can be used to engineer large protein-based nanostructures.…”

Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry

“…Supramolecular reactivation of inactivated enzymes have been studied on inactivated caspase-8 mutant and split-luciferase fragments. A mutated caspase-8 (D384A) featuring FGG motif at the N-terminus which shows enzymatically inactive towards its natural substrate caspase-3, could be fully reactivated upon addition of cucurbit[8]uril ( Dang et al, 2018 ). The FGG motif was applied to split-luciferase fragment pairs at the N-terminus that allowed cucurbit[8]uril to induce dimerization of luciferase and regenerate enzymatic activity ( Bosmans et al, 2016 ).…”

Selective Recognition of Amino Acids and Peptides by Small Supramolecular Receptors

“…Control over protein assembly and function is another route that is being explored for this supramolecular system [ 9 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 ]. Brunsveld and co-workers have used CB8 as a tool to aid in the dimerization of proteins, making use of the target Phe-Gly-Gly motif as an N -terminal modification, in order to better control enzyme activity [ 149 ] and protein assemblies [ 142 ].…”

“…A more recent study reported the role of CB8 as a modulator of enzymatic activity using caspase 8, an enzyme that is typically only active upon dimerization. In this work Dang et al, after observing that the Casp-8 N -terminal residues of the two monomers are in close proximity in the dimeric active state, engineered this region of the Casp-8 to have an Phe-Gly-Gly motif [ 149 ]. This motif allowed for the use of CB8 to control the dimerization in the inactive mutants and resulted in an easily modulated, dimeric form of Casp-8 active towards a synthetic ligand as well as its physiological substrate, caspase-3.…”