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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 wild-typeand V266H caspase-3 structures. (a) Caspase-3structure (PDB entry 2J30) highlighting β-strands 1–8 and active site loops L1–L4and L2′ in one protomer. The two boxes indicate regions ofmutations in the dimer interface and active sites. (b) H266 causesY195 to move toward T140 (helix 3) (PDB entry 4EHA). A salt bridgebetween K137 and E190 is also disrupted. Red spheres indicate watermolecules in WT caspase-3, and yellow spheres indicate water moleculesin caspase-3(V266H). The dashed lines indicate hydrogen bonds in wild-typecaspase-3, and the solid line indicates a hydrogen bond in caspase-3(V266H).(c) Comparison of active site residues F128, M61, F55, H121, and C163for WT and V266H proteins. For panels b and c, amino acids are coloredyellow for the V266H variant and gray for WT caspase-3.
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fig2: Comparison of wild-typeand V266H caspase-3 structures. (a) Caspase-3structure (PDB entry 2J30) highlighting β-strands 1–8 and active site loops L1–L4and L2′ in one protomer. The two boxes indicate regions ofmutations in the dimer interface and active sites. (b) H266 causesY195 to move toward T140 (helix 3) (PDB entry 4EHA). A salt bridgebetween K137 and E190 is also disrupted. Red spheres indicate watermolecules in WT caspase-3, and yellow spheres indicate water moleculesin caspase-3(V266H). The dashed lines indicate hydrogen bonds in wild-typecaspase-3, and the solid line indicates a hydrogen bond in caspase-3(V266H).(c) Comparison of active site residues F128, M61, F55, H121, and C163for WT and V266H proteins. For panels b and c, amino acids are coloredyellow for the V266H variant and gray for WT caspase-3.

Mentions: Conformational states of caspases. The procaspase monomerformsdimers either through folding and assembly, where the dimer is themost stable form (effector caspases, for example), through interactionwith death receptors (initiator caspases, for example), or throughthe action of kosmotropes (sodium citrate, for example). The dimerof the zymogen contains inactive and active conformations. Cleavageof the intersubunit linker (red loop) results in the formation oftwo active site loops, called L2 and L2′ (see Figure 2), colored green and red, respectively, and separationof the two subunits (large subunit, dark blue; small subunit, lightblue) to form the protomer. In the inactive zymogen-like caspase form,loop L2′ (green) remains bound in the dimer interface and theactive site loops are disordered. In the active caspase, L2′interacts with L2 of the opposite protomer and stabilizes the activeconformation. The equilibrium between the active and zymogen-likecaspase is affected by binding of substrate in the active site orallosteric inhibitor to the dimer interface (or other allosteric sites).As described here, the mature caspase ensemble also contains an inactiveconformer characterized by an intact substrate-binding pocket anddestabilized helix 3.


Modifying caspase-3 activity by altering allosteric networks.

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

Comparison of wild-typeand V266H caspase-3 structures. (a) Caspase-3structure (PDB entry 2J30) highlighting β-strands 1–8 and active site loops L1–L4and L2′ in one protomer. The two boxes indicate regions ofmutations in the dimer interface and active sites. (b) H266 causesY195 to move toward T140 (helix 3) (PDB entry 4EHA). A salt bridgebetween K137 and E190 is also disrupted. Red spheres indicate watermolecules in WT caspase-3, and yellow spheres indicate water moleculesin caspase-3(V266H). The dashed lines indicate hydrogen bonds in wild-typecaspase-3, and the solid line indicates a hydrogen bond in caspase-3(V266H).(c) Comparison of active site residues F128, M61, F55, H121, and C163for WT and V266H proteins. For panels b and c, amino acids are coloredyellow for the V266H variant and gray for WT caspase-3.
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Related In: Results  -  Collection

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Show All Figures
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fig2: Comparison of wild-typeand V266H caspase-3 structures. (a) Caspase-3structure (PDB entry 2J30) highlighting β-strands 1–8 and active site loops L1–L4and L2′ in one protomer. The two boxes indicate regions ofmutations in the dimer interface and active sites. (b) H266 causesY195 to move toward T140 (helix 3) (PDB entry 4EHA). A salt bridgebetween K137 and E190 is also disrupted. Red spheres indicate watermolecules in WT caspase-3, and yellow spheres indicate water moleculesin caspase-3(V266H). The dashed lines indicate hydrogen bonds in wild-typecaspase-3, and the solid line indicates a hydrogen bond in caspase-3(V266H).(c) Comparison of active site residues F128, M61, F55, H121, and C163for WT and V266H proteins. For panels b and c, amino acids are coloredyellow for the V266H variant and gray for WT caspase-3.
Mentions: Conformational states of caspases. The procaspase monomerformsdimers either through folding and assembly, where the dimer is themost stable form (effector caspases, for example), through interactionwith death receptors (initiator caspases, for example), or throughthe action of kosmotropes (sodium citrate, for example). The dimerof the zymogen contains inactive and active conformations. Cleavageof the intersubunit linker (red loop) results in the formation oftwo active site loops, called L2 and L2′ (see Figure 2), colored green and red, respectively, and separationof the two subunits (large subunit, dark blue; small subunit, lightblue) to form the protomer. In the inactive zymogen-like caspase form,loop L2′ (green) remains bound in the dimer interface and theactive site loops are disordered. In the active caspase, L2′interacts with L2 of the opposite protomer and stabilizes the activeconformation. The equilibrium between the active and zymogen-likecaspase is affected by binding of substrate in the active site orallosteric inhibitor to the dimer interface (or other allosteric sites).As described here, the mature caspase ensemble also contains an inactiveconformer characterized by an intact substrate-binding pocket anddestabilized 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