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Modifying caspase-3 activity by altering allosteric networks.

Cade C, Swartz P, MacKenzie SH, Clark AC - Biochemistry (2014)

Bottom Line: Mutations in presumed allosteric networks also decrease activity, although large structural changes are not observed.In contrast to the effects of small molecule allosteric regulators, the substrate-binding pocket is intact in the mutant, yet the enzyme is inactive.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry and ‡Center for Comparative Medicine and Translational Research, North Carolina State University , Raleigh, North Carolina 27695, United States.

ABSTRACT
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.

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Enzyme activityof wild-type caspase-3 and allosteric mutants.(a) kcat/KM values for each allosteric mutant. Full steady-state enzyme parametersare listed in Table 2. (b) Comparison of enzymeactivity relative to distance from H266, measured from α-carbons.The solid line is present only to show trends in the data.
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fig3: Enzyme activityof wild-type caspase-3 and allosteric mutants.(a) kcat/KM values for each allosteric mutant. Full steady-state enzyme parametersare listed in Table 2. (b) Comparison of enzymeactivity relative to distance from H266, measured from α-carbons.The solid line is present only to show trends in the data.

Mentions: Our previous studies of caspase-3(V266H)suggested that relieving steric clashes introduced by H266 shouldreturn activity to the enzyme.16 To testthis hypothesis, we introduced mutations atM61, F128, and Y195 in the context of H266, where each amino acidwas replaced with alanine. Tyrosine 195 was also replaced with phenylalanine(Table 1 and Figure 2). In addition, we replaced T140 with glycine to introduce a smalleramino acid into helix 3 and disrupt the hydrogen bond with Y195. Inthe V266H variant,16 M61 clashes with H121and disrupts hydrogen bondingbetween H121 and active site loop 1 (Figure 2c). Surface β-strands 4 and 5 (see Figure 2a) are observed to move relative to their positions in wild-typecaspase-3. The movement is exemplified by F128, which forms hydrophobiccontacts with M61 and F55 in wild-type caspase-3 (Figure 2c). In the V266H variant, F128 moves closer to theactive site, possibly causing M61 to clash with H121. The single mutantsM61A, F128A, T140G, Y195A, and Y195F were designed as controls forthe restorative double mutants, which have those same mutations inthe background of V266H. Data from enzyme activity assays show thatthe single mutations have little effect on activity (Figure 3a and Table 2). The activityof the F128A variant is the lowest of the control mutants, being reduced15-fold from that of the wild-type enzyme. Interestingly, the enzymeactivity of the Y195F variant is very high, ∼75% of the activityof wild-type caspase-3, demonstrating that the through-water hydrogenbond with T140 is not critical for stabilizing helix 3.


Modifying caspase-3 activity by altering allosteric networks.

Cade C, Swartz P, MacKenzie SH, Clark AC - Biochemistry (2014)

Enzyme activityof wild-type caspase-3 and allosteric mutants.(a) kcat/KM values for each allosteric mutant. Full steady-state enzyme parametersare listed in Table 2. (b) Comparison of enzymeactivity relative to distance from H266, measured from α-carbons.The solid line is present only to show trends in the data.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263430&req=5

fig3: Enzyme activityof wild-type caspase-3 and allosteric mutants.(a) kcat/KM values for each allosteric mutant. Full steady-state enzyme parametersare listed in Table 2. (b) Comparison of enzymeactivity relative to distance from H266, measured from α-carbons.The solid line is present only to show trends in the data.
Mentions: Our previous studies of caspase-3(V266H)suggested that relieving steric clashes introduced by H266 shouldreturn activity to the enzyme.16 To testthis hypothesis, we introduced mutations atM61, F128, and Y195 in the context of H266, where each amino acidwas replaced with alanine. Tyrosine 195 was also replaced with phenylalanine(Table 1 and Figure 2). In addition, we replaced T140 with glycine to introduce a smalleramino acid into helix 3 and disrupt the hydrogen bond with Y195. Inthe V266H variant,16 M61 clashes with H121and disrupts hydrogen bondingbetween H121 and active site loop 1 (Figure 2c). Surface β-strands 4 and 5 (see Figure 2a) are observed to move relative to their positions in wild-typecaspase-3. The movement is exemplified by F128, which forms hydrophobiccontacts with M61 and F55 in wild-type caspase-3 (Figure 2c). In the V266H variant, F128 moves closer to theactive site, possibly causing M61 to clash with H121. The single mutantsM61A, F128A, T140G, Y195A, and Y195F were designed as controls forthe restorative double mutants, which have those same mutations inthe background of V266H. Data from enzyme activity assays show thatthe single mutations have little effect on activity (Figure 3a and Table 2). The activityof the F128A variant is the lowest of the control mutants, being reduced15-fold from that of the wild-type enzyme. Interestingly, the enzymeactivity of the Y195F variant is very high, ∼75% of the activityof wild-type caspase-3, demonstrating that the through-water hydrogenbond with T140 is not critical for stabilizing helix 3.

Bottom Line: Mutations in presumed allosteric networks also decrease activity, although large structural changes are not observed.In contrast to the effects of small molecule allosteric regulators, the substrate-binding pocket is intact in the mutant, yet the enzyme is inactive.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry and ‡Center for Comparative Medicine and Translational Research, North Carolina State University , Raleigh, North Carolina 27695, United States.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus