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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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Comparison of helix 3 mutants. (a and b) Crystal structureof T140Vcomparing the dimer interface (a) and active site (b) with WT. Aminoacids are colored yellow for the mutant and gray for WT caspase-3.Red spheres represent conserved water molecules in the interface,and the dashed lines represent the H-bonding network. (c and d) MDsimulations of T140V. Two hundred frames (at 250 ps intervals) ofthe 50 ns simulation demonstrate movements of the amino acids in helix3 (c) (left panel, protomer A; right panel, protomer B) and the dimerinterface (d). For the sake of clarity, K138 is colored gray and bluewhile K137 is colored yellow and blue (c). Dimer interface (e) andactive site (f) of T140F. In panel e, red spheres represent conservedwater molecules in the interface. For panel f, amino acids are coloredyellow for the mutant and gray for WT caspase-3.
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fig7: Comparison of helix 3 mutants. (a and b) Crystal structureof T140Vcomparing the dimer interface (a) and active site (b) with WT. Aminoacids are colored yellow for the mutant and gray for WT caspase-3.Red spheres represent conserved water molecules in the interface,and the dashed lines represent the H-bonding network. (c and d) MDsimulations of T140V. Two hundred frames (at 250 ps intervals) ofthe 50 ns simulation demonstrate movements of the amino acids in helix3 (c) (left panel, protomer A; right panel, protomer B) and the dimerinterface (d). For the sake of clarity, K138 is colored gray and bluewhile K137 is colored yellow and blue (c). Dimer interface (e) andactive site (f) of T140F. In panel e, red spheres represent conservedwater molecules in the interface. For panel f, amino acids are coloredyellow for the mutant and gray for WT caspase-3.

Mentions: The crystal structure of the T140V variant shows that the six conservedwater molecules are presentin the dimer interface, as with wild-type caspase-3 (Figure 7a). The side chain of Y195 is shifted somewhat towardhelix 3, but it is closer to the position observed in wild-type caspase-3than that of the V266H variant. At the site of the mutation, helix3 overlays well with the comparable helix in wild-type caspase-3.The salt bridge between K137 and E190 is intact, but a through-waterH-bond between Y195 and T140 is missing in the mutant (data not shown).In the active site, the side chain of M61 is rotated so that the terminalCH3 is oriented toward solvent, but F128, F55, H121, andC163 overlay well with the wild-type active site (Figure 7b). Molecular dynamics simulations of this mutantshow that K137 is very mobile on one side of the interface, whileK137′ is less mobile and remains close to E190 for longer times(data not shown). Helix 3 is less stable than observed in wild-typecaspase-3 but does not fully undergo the transition to the inactiveconformation (Figure 7c). Although K137 interactswith the carbonyl of P201′ across the interface and K138 rotatestoward E190, the movements are transient such that the helical structureis mostly favored. During the helix fluctuation, the side chain ofY195 rotates approximately 90°, althoughthe hydroxyl retains its orientation toward helix 3 (Figure 7d).


Modifying caspase-3 activity by altering allosteric networks.

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

Comparison of helix 3 mutants. (a and b) Crystal structureof T140Vcomparing the dimer interface (a) and active site (b) with WT. Aminoacids are colored yellow for the mutant and gray for WT caspase-3.Red spheres represent conserved water molecules in the interface,and the dashed lines represent the H-bonding network. (c and d) MDsimulations of T140V. Two hundred frames (at 250 ps intervals) ofthe 50 ns simulation demonstrate movements of the amino acids in helix3 (c) (left panel, protomer A; right panel, protomer B) and the dimerinterface (d). For the sake of clarity, K138 is colored gray and bluewhile K137 is colored yellow and blue (c). Dimer interface (e) andactive site (f) of T140F. In panel e, red spheres represent conservedwater molecules in the interface. For panel f, amino acids are coloredyellow for the mutant and gray for WT caspase-3.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Comparison of helix 3 mutants. (a and b) Crystal structureof T140Vcomparing the dimer interface (a) and active site (b) with WT. Aminoacids are colored yellow for the mutant and gray for WT caspase-3.Red spheres represent conserved water molecules in the interface,and the dashed lines represent the H-bonding network. (c and d) MDsimulations of T140V. Two hundred frames (at 250 ps intervals) ofthe 50 ns simulation demonstrate movements of the amino acids in helix3 (c) (left panel, protomer A; right panel, protomer B) and the dimerinterface (d). For the sake of clarity, K138 is colored gray and bluewhile K137 is colored yellow and blue (c). Dimer interface (e) andactive site (f) of T140F. In panel e, red spheres represent conservedwater molecules in the interface. For panel f, amino acids are coloredyellow for the mutant and gray for WT caspase-3.
Mentions: The crystal structure of the T140V variant shows that the six conservedwater molecules are presentin the dimer interface, as with wild-type caspase-3 (Figure 7a). The side chain of Y195 is shifted somewhat towardhelix 3, but it is closer to the position observed in wild-type caspase-3than that of the V266H variant. At the site of the mutation, helix3 overlays well with the comparable helix in wild-type caspase-3.The salt bridge between K137 and E190 is intact, but a through-waterH-bond between Y195 and T140 is missing in the mutant (data not shown).In the active site, the side chain of M61 is rotated so that the terminalCH3 is oriented toward solvent, but F128, F55, H121, andC163 overlay well with the wild-type active site (Figure 7b). Molecular dynamics simulations of this mutantshow that K137 is very mobile on one side of the interface, whileK137′ is less mobile and remains close to E190 for longer times(data not shown). Helix 3 is less stable than observed in wild-typecaspase-3 but does not fully undergo the transition to the inactiveconformation (Figure 7c). Although K137 interactswith the carbonyl of P201′ across the interface and K138 rotatestoward E190, the movements are transient such that the helical structureis mostly favored. During the helix fluctuation, the side chain ofY195 rotates approximately 90°, althoughthe hydroxyl retains its orientation toward helix 3 (Figure 7d).

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