Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.

Modifying caspase-3 activity by altering allosteric networks.

Caspases have several allosteric sites that bind small molecules or peptides. Allosteric regulators are known to affect caspase enzyme activity, in general, by facilitating large conformational changes that convert the active enzyme to a zymogen-like form in which the substrate-binding pocket is disordered. Mutations in presumed allosteric networks also decrease activity, although large structural changes are not observed. Mutation of the central V266 to histidine in the dimer interface of caspase-3 inactivates the enzyme by introducing steric clashes that may ultimately affect positioning of a helix on the protein surface. The helix is thought to connect several residues in the active site to the allosteric dimer interface. In contrast to the effects of small molecule allosteric regulators, the substrate-binding pocket is intact in the mutant, yet the enzyme is inactive. We have examined the putative allosteric network, in particular the role of helix 3, by mutating several residues in the network. We relieved steric clashes in the context of caspase-3(V266H), and we show that activity is restored, particularly when the restorative mutation is close to H266. We also mimicked the V266H mutant by introducing steric clashes elsewhere in the allosteric network, generating several mutants with reduced activity. Overall, the data show that the caspase-3 native ensemble includes the canonical active state as well as an inactive conformation characterized by an intact substrate-binding pocket, but with an altered helix 3. The enzyme activity reflects the relative population of each species in the native ensemble.

Slow folding and assembly of a procaspase-3 interface variant.

Caspases execute apoptosis and exist in the cell as inactive zymogens (procaspases) prior to activation. Initiator procaspases are monomers that must dimerize for activation, while effector procaspases, such as procaspase-3, are stable dimers that must be processed for activation. The dimer interface regions of the two subfamilies are different, although the role of the interface in oligomerization is not known. Equilibrium and kinetic folding studies were performed on procaspase-3(C163S,V266H), an interface variant, to determine the importance of the dimer interface in the folding of procaspase-3. Equilibrium folding data at pH 5 and 7 display a hysteresis, indicating a kinetically controlled folding reaction. Refolding kinetic studies reveal a complex burst phase, followed by a series of monomeric intermediates. At longer refolding times, the monomer populates a species that becomes kinetically trapped and slowly aggregates. Unfolding kinetic studies reveal a hyperfluorescent native ensemble that unfolds to form highly structured monomeric intermediates that unfold very slowly. Dimerization is very slow, likely because of the inability to correctly orient the histidine residues in the interface, so the initial encounter complex for dimerization is inefficient. As a consequence, the monomer folds into species that aggregate. Introducing a histidine into the interface of procaspase-3 prevents activation by acting as a negative design element, providing evidence that the interface region is a site of regulation of caspase assembly in general by affecting the rate of dimerization.

Lengthening the intersubunit linker of procaspase 3 leads to constitutive activation.

The conformational ensemble of procaspase 3, the primary executioner in apoptosis, contains two major forms, inactive and active, with the inactive state favored in the native ensemble. A region of the protein known as the intersubunit linker (IL) is cleaved during maturation, resulting in movement of the IL out of the dimer interface and subsequent active site formation (activation-by-cleavage mechanism). We examined two models for the role of the IL in maintaining the inactive conformer, an IL-extension model versus a hydrophobic cluster model, and we show that increasing the length of the IL by introducing 3-5 alanines results in constitutively active procaspases. Active site labeling and subsequent analyses by mass spectrometry show that the full-length zymogen is enzymatically active. We also show that minor populations of alternately cleaved procaspase result from processing at D169 when the normal cleavage site, D175, is unavailable. Importantly, the alternately cleaved proteins have little to no activity, but increased flexibility of the linker increases the exposure of D169. The data show that releasing the strain of the short IL, in and of itself, is not sufficient to populate the active conformer of the native ensemble. The IL must also allow for interactions that stabilize the active site, possibly from a combination of optimal length, flexibility in the IL, and specific contacts between the IL and interface. The results provide further evidence that substantial energy is required to shift the protein to the active conformer. As a result, the activation-by-cleavage mechanism dominates in the cell.

Death by caspase dimerization.

Controlled cell death, or apoptosis, occurs in response to many different environmental stimuli. The apoptotic cascade that occurs within the cell in response to these cues leads to morphological and biochemical changes that trigger the dismantling and packaging of the cell. Caspases are a family of cysteine-dependent aspartate-directed proteases that play an integral role in the cascade that leads to apoptosis. Caspases are grouped as either initiators or effectors of apoptosis, depending on where they enter the cell death process. Prior to activation, initiator caspases are present as monomers that must dimerize for full activation whereas effector caspases are present as dimeric zymogens that must be processed for full activation. The stability of the dimer may be due predominately to the interactions in the dimer interface as each caspase has unique properties in this region that lend to its specific mode of activation. Moreover, dimerization is responsible for active site formation because both monomers contribute residues that enable the formation of a fully functional active site. Overall, dimerization plays a key role in the ability of caspases to form fully functional proteases.

Phage display and structural studies reveal plasticity in substrate specificity of caspase-3a from zebrafish.

The regulation of caspase-3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase-3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase-3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5-P1' or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X-ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub-site by increasing flexibility of one active site loop and by affecting hydrogen-bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase-6 in zebrafish development.

Redesigning the procaspase-8 dimer interface for improved dimerization.

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.

A bifunctional allosteric site in the dimer interface of procaspase-3.

The dimer interface of caspase-3 contains a bifunctional allosteric site in which the enzyme can be activated or inactivated, depending on the context of the protein. In the mature caspase-3, the binding of allosteric inhibitors to the interface results in an order-to-disorder transition in the active site loops. In procaspase-3, by contrast, the binding of allosteric activators to the interface results in a disorder-to-order transition in the active site. We have utilized the allosteric site to identify a small molecule activator of procaspase and to characterize its binding to the protease. The data suggest that an efficient activator must stabilize the active conformer of the zymogen by expelling the intersubunit linker from the interface, and it must interact with active site residues found in the allosteric site. Small molecule activators that fulfill the two requirements should provide scaffolds for drug candidates as a therapeutic strategy for directly promoting procaspase-3 activation in cancer cells.

The potential for caspases in drug discovery.

Caspases are a family of proteases that are involved in the execution of apoptosis and the inflammatory response. A plethora of diseases occur as a result of the dysregulation of apoptosis and inflammation, and caspases have been targeted as a therapeutic strategy to halt the progression of such diseases. Hundreds of peptide and peptidomimetic inhibitors have been designed and tested, but only a few have advanced to clinical trials because of poor drug-like properties and pharmacological constraints. Although much effort has been focused on inhibiting caspases, there are many diseases that result from a decrease in apoptosis, thus activating procaspases could also be a viable therapeutic strategy. To this end, recent efforts have focused on the design of procaspase-3 activators. This review highlights the current progress in the rational design of both specific and pan-caspase inhibitors, as well as procaspase-3 activators.

Targeting cell death in tumors by activating caspases.

Cytotoxic approaches to killing tumor cells, such as chemotherapeutic agents, gamma-irradiation, suicide genes or immunotherapy, have been shown to induce cell death through apoptosis. The intrinsic apoptotic pathway is activated following treatment with cytotoxic drugs, and these reactions ultimately lead to the activation of caspases, which promote cell death in tumor cells. In addition, activation of the extrinsic apoptotic pathway with death-inducing ligands leads to an increased sensitivity of tumor cells toward cytotoxic stimuli, illustrating the interplay between the two cell death pathways. In contrast, tumor resistance to cytotoxic stimuli may be due to defects in apoptotic signaling. As a result of their importance in killing cancer cells, a number of apoptotic molecules are implicated in cancer therapy. The knowledge gleaned from basic research into apoptotic pathways from cell biological, structural, biochemical, and biophysical approaches can be used in strategies to develop novel compounds that eradicate tumor cells. In addition to current drug targets, research into molecules that activate procaspase-3 directly may show the direct activation of the executioner caspase to be a powerful therapeutic strategy in the treatment of many cancers.