Redesigning the procaspase‐8 dimer interface for improved dimerization

Abstract

Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.

Caspase Allostery and Conformational Selection

“…This hypothesis was tested in two ways. First, a procaspase-3 variant was examined for changes in dimerization, where a mutation was introduced into the dimer interface. , Second, the dimer interface of caspase-8, which forms weak dimers in solution, ,,, was mutated to improve dimer formation …”

“…Although the negative design elements may decrease dimerization efficiency, they also may be important to lower aggregation of proteins with an exposed β-sheet, as might occur in the caspase protomer, due to the requirement for more precise complementarity. Mutations of several negative design elements in caspase-8 resulted in improved dimerization, although the variants did not form constitutive dimers . Together with the experiments of procaspase-3, however, the data show that caspases have inefficient dimerization encounter complexes, and mutations that further decrease the efficiency of dimerization result in effectively trapping the protein in the monomeric state.…”

Heterogeneous responses to low level death receptor activation are explained by random molecular assembly of the Caspase-8 activation platform

“…The dimeric ProCasp8 association-dissociation balance has been suggested to play a crucial role in the molecular control of apoptotic responses after DR activation [21]. However, as demonstrated by mutagenesis studies, ProCasp8 dimerization alone is not sufficient to enhance apoptotic responses in vivo [22]. Instead, formation of the DISC or RIPoptosome platforms are necessary for effective ProCasp8 dimerization and Casp8 activation [10,23,24].…”

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

“…Fusion of casp‐8 to a FKBP‐rapamycin associated protein domain allowed a dimeric FK506 small molecule analogue to induce dimerization of this fusion construct, resulting in increased casp‐8 activation . Redesigned casp‐8 dimer interfaces enable improved dimerization, while not enhancing apoptosis . Structural and biochemical studies on casp‐8 have provided new insights through a fully monomeric engineered casp‐8, which features abolished or, interestingly, only reduced enzymatic activity .…”