Modifying Caspase-3 Activity by Altering Allosteric Networks

Cade, C.; Swartz, Paul; MacKenzie, S.H.; Clark, A. Clay

doi:10.1021/bi500874k

Cited by 27 publications

(33 citation statements)

References 41 publications

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“…Although poorly understood, the phosphorylation of caspase-3 in the C-terminal loop of helix-3 is of particular interest because it is thought to represent a common allosteric mechanism in all caspases (25,26). In addition, T152 is part of a hydrophobic cluster of amino acids that stabilize the active site loops across the dimer interface.…”

Section: Discussionmentioning

confidence: 99%

“…One allosteric site of interest, S150, is located in a loop at the C-terminal end of helix-3 near the dimer interface, and this serine residue (or threonine in caspase-7) is conserved in all human caspases except caspases-10 and -14 (4). Helix-3 is an important regulator of effector caspase activity because fluctuations in the Nterminal region of the helix disrupt conserved water networks in caspase-3 and reposition the catalytic cysteine and histidine (24,25). In addition, the same region of helix-3 and the adjoining β-strands undergo a coil-to-helix transition in caspase-6, which also disrupts the catalytic residues by extending helix-3 (22).…”

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confidence: 99%

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Modifications to a common phosphorylation network provide individualized control in caspases

Thomas

Grinshpon

Swartz

et al. 2018

Journal of Biological Chemistry

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Caspase-3 activation and function has been well defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation.Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, S150 evolved with the apoptotic caspases, while T152 is a more recent evolutionary event in mammalian caspase-3. Substitutions at S150 result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of T152 in the conserved loop introduces a "kill switch" in mammalian caspase-3 while the more ancient S150 reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in both a common pathway for lowering activity during development as well as introducing a more recent cluster-specific switch to abolish activity.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Modifications to a common phosphorylation network provide individualized control in caspases

Thomas

Grinshpon

Swartz

et al. 2018

Journal of Biological Chemistry

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“…binding to the active or allosteric sites affects the partitioning of active vs. inactive conformations, so the activity of the caspase reports on the relative distribution of active and inactive states within the ensemble (23,25), providing a mechanism to fine-tune activity. An allosteric site in the dimer interface ( Fig.…”

Section: Significancementioning

confidence: 99%

“…1B, state 2), whereas caspase-1, -6, and -7 contain low-energy, so-called "closed-loop" inactive states with disordered active-site loops (Fig. 1B, state 3) (20)(21)(22)(23)(24)(25). Conformational selection by ligand…”

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confidence: 99%

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

Maciag

MacKenzie

Tucker

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 highresolution (<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.C aspase function in cell development and cell death results from a continuum of enzyme activity, in which an as-yetundefined activity threshold is required for cell death. At subthreshold levels, caspase activity is important for a variety of physiological reactions (referred to as adaptive responses), including remodeling the cytoplasm (1), cell differentiation (2), neuron pruning (3), receptor endocytosis (4), macrophage function (5), and development of the eye lens (6) and inner ear (7). The roles of caspases in apoptosis are well known, but their roles in adaptive responses are less clear, particularly in regard to how cells set the threshold of caspase activity to limit apoptosis while ensuring sufficient activity for signaling and differentiation.Cells use two general mechanisms to modify caspase activity, through modulating levels of active caspase or through allosteric mechanisms that change the distribution of conformations in the native ensemble, although the two are not mutually exclusive. Levels of caspase-3 are controlled by cleavage of the inactive zymogen to yield a dimer of protomers (Fig. 1A) (8,9), and this process is responsive to several signaling pathways, such as transient expression of the Bad-Bax cascade (10) or phosphorylation of the zymogen (Fig. 1B) (11). Alternatively, inhibitor of apoptosis proteins (IAPs) affect levels of active caspase-3 by dire...

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“…In fact, a recent study demonstrated that the catalytic activity of mature caspase-3 is also enhanced by the structural perturbation as well as that of uncleavable procaspase-3s. 27 Namely, we should differentiate effects of a bioactive synthetic molecule on zymogens, their mature enzymes or both of them by individually investigating effects of the synthetic molecule on these proteins. Accordingly, this paper focuses on the possibility of effects of 1 on mature caspase-3.…”

Section: ¹1mentioning

confidence: 99%

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

Matsuo

Yamada

Ishida

et al. 2015

Bulletin of the Chemical Society of Japan

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Caspase-3, an apoptosis-executing enzyme with a dimeric structure, and its zymogen (procaspase-3) are target proteins in cancer chemotherapy using synthetic compounds. Previous work has shown that PAC-1 (1st procaspaseactivating compound) affects the activation of procaspase-3 in cancerous cells through the Zn 2+ -chelation mechanism. However, we found that the compound also enhances the mature caspase-3 activity in the absence of Zn 2+. The enhancement of the mature caspase-3 activity by PAC-1 followed a MichaelisMenten-type saturation dependency with respect to the concentration of the compound. PAC-1 induced the change in the UVvis spectrum of the mature caspase-3. From these findings, we propose a direct structural interaction of PAC-1 with the protein core. According to a molecular dynamics calculation for the complex of PAC-1 and mature caspase-3, PAC-1 is likely to bind to the interface between the dimeric structure in mature caspase-3. The interactions between PAC-1 and amino acid residues that affect the catalytic activity of mature caspase-3 are suggested. Throughout this work, diverse functions of the apoptosis-inducing compound are disclosed.

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Modifying Caspase-3 Activity by Altering Allosteric Networks

Cited by 27 publications

References 41 publications

Modifications to a common phosphorylation network provide individualized control in caspases

Modifications to a common phosphorylation network provide individualized control in caspases

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

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

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