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

Walters J, Pop C, Scott FL, Drag M, Swartz P, Mattos C, Salvesen GS, Clark AC - Biochem. J. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Our previous biochemical data for D3A,V266E [11], as well as that provided in the present study, show that the V266E mutation effectively shifts the zymogen to an active conformer. Currently there are no structures for procaspase-3, so we generated homology models of the putative active and inactive zymogen in order to examine the conformational switch. Owing to their high sequence identity, we modelled inactive WT after procaspase-7 [35,36], and energy minimized the structures as described in the Experimental section (and the Supplementary online data) to assure that our final model is energetically feasible. We find that, similar to procaspase-7, the active sites in our model of the inactive zymogen are disorganized and the IL occupies the central cavity. There are two monomers, represented by chains A and B, forming a complete dimer in the asymmetric unit of the procaspase-7 X-ray crystal structure. Our model of the inactive procaspase-3 is similar to the entire dimer. Although the IL from chain A (IL-A) in the procaspase-7 template mostly remains exposed to solvent, a large portion of IL-B is buried in the interface, making contacts with several interface residues and shielding the hydrophobic core from water (Figure 5A).


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

Walters J, Pop C, Scott FL, Drag M, Swartz P, Mattos C, Salvesen GS, Clark AC - Biochem. J. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2805924&req=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.
Mentions: Our previous biochemical data for D3A,V266E [11], as well as that provided in the present study, show that the V266E mutation effectively shifts the zymogen to an active conformer. Currently there are no structures for procaspase-3, so we generated homology models of the putative active and inactive zymogen in order to examine the conformational switch. Owing to their high sequence identity, we modelled inactive WT after procaspase-7 [35,36], and energy minimized the structures as described in the Experimental section (and the Supplementary online data) to assure that our final model is energetically feasible. We find that, similar to procaspase-7, the active sites in our model of the inactive zymogen are disorganized and the IL occupies the central cavity. There are two monomers, represented by chains A and B, forming a complete dimer in the asymmetric unit of the procaspase-7 X-ray crystal structure. Our model of the inactive procaspase-3 is similar to the entire dimer. Although the IL from chain A (IL-A) in the procaspase-7 template mostly remains exposed to solvent, a large portion of IL-B is buried in the interface, making contacts with several interface residues and shielding the hydrophobic core from water (Figure 5A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.

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.

Show MeSH
Related in: MedlinePlus