Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Dec 10;424(3):335-45.
doi: 10.1042/BJ20090825.

A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis

Jad Walters  1 , Cristina Pop, Fiona L Scott, Marcin Drag, Paul Swartz, Carla Mattos, Guy S Salvesen, A Clay Clark
Affiliations

A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis

Jad Walters et al. Biochem J. .

Abstract

The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 A (1 A = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Procaspase-3(D3A,V266E) is enzymatically active without cleavage of the IL
(A) The interface mutation V266E was designed in the context of wild-type caspase-3 (WT) and the uncleavable procaspase-3(D9A,D28A,D175A), called D3A. Low expression generates ‘one-chain’ procaspase-3 (Pro-WT). Overexpression generates the ‘two-chain’ caspase-3 (WT or V266E) by automaturation. ‘Pro’ refers to the pro-domain. (B) Structure of WT caspase-3 (PDB code 2J30) highlighting the active site loops L1 (yellow), L2 (red), L3 (blue), L4 (brown) and L2′ (cyan). The prime (′) indicates residues from the second monomer. For clarity, only one active site is labelled. (C) Labelling the V266E mutants by affinity-based probes. Proteins labelled with bEVD-AOMK were probed by Western blot analysis with anti-biotin, anti-cleaved caspase-3, anti-full-length caspase-3 or anti-His-tag antibodies, or subjected to trichloroacetic acid precipitation and stained with Coomassie Blue. The positive control was WT, and the negative control was Pro-WT. The asterisk indicates a contaminating protein from E. coli expression. Molecular masses are shown to the left in kDa. (D) Determining the substrate specificity of the recombinant caspase-3 mutants. Activity of the caspase-3 mutants (10 nM) was measured using a tetrapeptide positional scanning library, with P1 fixed as an aspartate residue and 7-amino-4-carbamoylmethylcoumarin as the fluorophore group. Hydrolysis rates are presented as a percentage of the maximum rate for each subset (P2, P3 and P4).
Figure 2
Figure 2. V266E mutants kill cells more efficiently than does the wild-type caspase-3
(A) HEK-293A cells were transiently transfected with FLAG-tagged caspase-3 DNA (or 50 ng Bax/0.95 μg empty vector), and Annexin V-positive cells were quantified after 24 h. Z-VAD-FMK (100 μM) or DMSO was added to the cultures 2 h post-transfection. The values represent the means for three independent experiments±S.D. (B) Western blots of the cellular lysates from (A) against anti-full-length caspase-3 or anti-FLAG antibodies, for detection of the transfected constructs, or anti-cleaved PARP. The lower panel shows the loading control of Hsp90.
Figure 3
Figure 3. Wild-type caspase-3 lysates display higher DEVD-ase activity than those of V266E interface mutants
(A) Lysates from cells transfected for 24 h with the indicated plasmids were assayed for enzymatic activity. The values represent the means ± S.D. for three independent experiments. Values for the mean percentage apoptotic death are from Figure 2(A). RFU, relative fluorescence units. (B) Caspase-3 mutants (300 pM) were incubated with XIAP at the indicated concentration in XIAP assay buffer (see Supplementary Experimental section) for 30 min at 37 °C, and the remaining activity was tested against Ac-DEVD-AFC (100 μM). The activity rates were plotted as the percentage of the maximum velocity in the absence of the inhibitor. The data were fitted to an equation describing the enzymatic activity in the presence of a reversible competitive inhibitor (see Supplementary Experimental section), and results of the fits are shown in Table 1.
Figure 4
Figure 4. V266E changes the dimer interface
(A) Interactions in L2′ (cyan) with L4 (brown) and the P4-binding site of WT. Inset: N-termini of L2′ for V266E begins at Lys186. (B) Dimer interface of WT (upper panel) demonstrating neutralization of the positive charge of Arg164 and six conserved water molecules (see Supplementary online data), and dimer interface of V266E (lower panel). Contacts between Glu124 and Arg164 are maintained in V266E, but two water molecules are removed and two are displaced by the carboxylate of Glu266. (C) Comparison of the dimer interface of V266E (grey) with that of caspase-1 (yellow). The rotamer at Glu390 in caspase-1 allows closer contact with the active site arginine. (D) V266E modelled using the rotamer found in caspase-1 demonstrates intra- and inter-subunit steric clashes with Tyr197 and Glu266′.
Figure 5
Figure 5. Homology models of (in)active procaspase-3
(A) Model of inactive procaspase-3 demonstrating binding of the IL (cyan) in the dimer interface, preventing organization of the active site loops. Colour code is the same as that used in Figure 1(B). Note that L2 and L2′ are covalently connected in the IL. (B) Superposition of inactive procaspase-3 (green residues) and of procaspase-7 (yellow) shows the blocking segment of IL-B, residues 184′–189′ (procaspase-3 numbering), prevents insertion of active site loop 3 from monomer A. For clarity, only one residue from the blocking segment of procaspase-7 is shown (semi-transparent sticks, Tyr211), whereas all of the residues of the blocking segment of procaspase-3 are highlighted (green). Upon cleavage of the IL, L2′, where the blocking segment resides, rotates ~ 180 ° and vacates the interface. Subsequently, a portion of the substrate-binding loop is permitted to insert in the interface. Arg164, Tyr197 and Pro201 engage in a stacking interaction (shown as the white residues) once L3 inserts in the interface. Insertion of the substrate-binding loop is permitted in the active procaspase-3 (blue ribbon) as the blocking segment lifts out of the interface upon activation (blue residues). (C) Superposition of procaspase-7 (yellow) and of inactive procaspase-3 (green) reveals a blocking segment involving residues 179′–180′ (caspase-3 numbering) of IL-B and Val189 of IL-A, preventing insertion of L3 in the active site of monomer B. L3 (white ribbon, WT) cannot insert into the interface until the blocking segment (green, inactive procaspase-3; yellow, procaspase-7) vacates the interface.
Figure 6
Figure 6. V266E expels the IL from the interface
(A) Hydrophobic cluster in the inactive procaspase-3 centred about Val266. Residues in the IL (cyan) of both monomers contribute to the cluster. (B) Because the glutamate side chain is longer than that of valine, the V266E mutation probably disrupts the hydrophobic cluster, preventing the IL from binding in the interface. (C) Comparison of the IL in the inactive and active procaspase-3. H-bonds contributed by the loop bundle, and centred about Asp169, are critical to active site stabilization. These contacts form only in the active conformer.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Boatright K. M., Salvesen G. S. Mechanisms of caspase activation. Curr. Opin. Cell Biol. 2003;15:725–731. - PubMed
    1. Renatus M., Stennicke H. R., Scott F. L., Liddington R. C., Salvesen G. S. Dimer formation drives the activation of the cell death protease caspase 9. Proc. Natl. Acad. Sci. U.S.A. 2001;98:14250–14255. - PMC - PubMed
    1. Pop C., Fitzgerald P., Green D. R., Salvesen G. S. Role of proteolysis in caspase-8 activation and stabilization. Biochemistry. 2007;46:4398–4407. - PubMed
    1. Pop C., Chen Y.-R., Smith B., Bose K., Bobay B., Tripathy A., Franzen S., Clark A. C. Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer. Biochemistry. 2001;40:14224–14235. - PubMed
    1. Bose K., Pop C., Feeney B., Clark A. C. An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3. Biochemistry. 2003;42:12298–12310. - PMC - PubMed

Publication types

MeSH terms

Associated data