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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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Structures and MD simulationsof steric clash and salt bridge mutants.Comparison of active site residues (a) and of active site loop 1 (b)from the crystal structure of F55W. In panel a, amino acids are coloredyellow for the mutant and gray for WT caspase-3. The new hydrogenbond between W55 and G129 is shown by the dashed line. In panel b,a lack of electron density (black mesh) in active site 1 shows disorderin several residues of loop 1 (left), whereas the loop is well orderedin the second active site (right). MD simulations show movements inthe active site residues (c) and helix 3 (d) of F55W. (e and f) Comparisonof helix 3 for K137A (left) and E190A (right) from the crystal structures(e) or from MD simulations (f). In panel e, the green sphere representsa new water molecule observed in the mutants. Amino acids are coloredyellow for the mutant and gray for WT caspase-3. For panels c, d,and f, 200 frames (at 250 ps intervals) of the 50 ns simulations areshown to demonstrate movements of the amino acids.
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fig8: Structures and MD simulationsof steric clash and salt bridge mutants.Comparison of active site residues (a) and of active site loop 1 (b)from the crystal structure of F55W. In panel a, amino acids are coloredyellow for the mutant and gray for WT caspase-3. The new hydrogenbond between W55 and G129 is shown by the dashed line. In panel b,a lack of electron density (black mesh) in active site 1 shows disorderin several residues of loop 1 (left), whereas the loop is well orderedin the second active site (right). MD simulations show movements inthe active site residues (c) and helix 3 (d) of F55W. (e and f) Comparisonof helix 3 for K137A (left) and E190A (right) from the crystal structures(e) or from MD simulations (f). In panel e, the green sphere representsa new water molecule observed in the mutants. Amino acids are coloredyellow for the mutant and gray for WT caspase-3. For panels c, d,and f, 200 frames (at 250 ps intervals) of the 50 ns simulations areshown to demonstrate movements of the amino acids.

Mentions: The enzyme activity data showedthat increasing the size of theside chain at position 55 in loop 1 correlated with a decrease inactivity (Table 2). Unfortunately, we are unableto report the structure of the F55Y variant, but we determined thestructure of F55W to 1.9 Å resolution (Table S3 of the Supporting Information). The active site groupsoverlay well with those of wild-type caspase-3with the exception of M61, which is displaced because of the largetryptophan side chain. In addition, the indole nitrogen forms a newH-bond with the carbonyl of G129, which resides on a turn betweenβ-strands 4 and 5 (Figure 8a). Whilethe electron density is good for W55 in both active sites, severalresidues in loop 1 (H56–T62) are disordered in one active site,indicating that the H-bond between the catalytic H121 and the backbonecarbonyl of T62 is disrupted (Figure 8b). Inthe interface, the six conserved water molecules are present in themutant, and the interface overlays very closely with that of wild-typecaspase-3 (Figure S6d of the Supporting Information). MD simulations of the F55W variant show that the H-bond betweenW55 and G129 is maintained throughout the simulation, resulting ina lower mobility of M61 (Figure 8c). In oneactive site, the side chain of H121 rotates toward C163, as describedabove for other mutants (Figure 8c, left panel).In the second active site, however, H121 remains H-bonded to the carbonylof T62 in loop 1; that is, it does not rotate toward C163 (Figure 8c, right panel). In both active sites, M61 remainsin close contact with the hydrophobic cluster of F128 and W55 (Figure 8C). Finally, there are no major changes in the dimerinterface (Figure S6d of the Supporting Information), and helix 3 remains stable, as shown by the lack of rotation inK137 and K138 (Figure 8d).


Modifying caspase-3 activity by altering allosteric networks.

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

Structures and MD simulationsof steric clash and salt bridge mutants.Comparison of active site residues (a) and of active site loop 1 (b)from the crystal structure of F55W. In panel a, amino acids are coloredyellow for the mutant and gray for WT caspase-3. The new hydrogenbond between W55 and G129 is shown by the dashed line. In panel b,a lack of electron density (black mesh) in active site 1 shows disorderin several residues of loop 1 (left), whereas the loop is well orderedin the second active site (right). MD simulations show movements inthe active site residues (c) and helix 3 (d) of F55W. (e and f) Comparisonof helix 3 for K137A (left) and E190A (right) from the crystal structures(e) or from MD simulations (f). In panel e, the green sphere representsa new water molecule observed in the mutants. Amino acids are coloredyellow for the mutant and gray for WT caspase-3. For panels c, d,and f, 200 frames (at 250 ps intervals) of the 50 ns simulations areshown to demonstrate movements of the amino acids.
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Related In: Results  -  Collection

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

fig8: Structures and MD simulationsof steric clash and salt bridge mutants.Comparison of active site residues (a) and of active site loop 1 (b)from the crystal structure of F55W. In panel a, amino acids are coloredyellow for the mutant and gray for WT caspase-3. The new hydrogenbond between W55 and G129 is shown by the dashed line. In panel b,a lack of electron density (black mesh) in active site 1 shows disorderin several residues of loop 1 (left), whereas the loop is well orderedin the second active site (right). MD simulations show movements inthe active site residues (c) and helix 3 (d) of F55W. (e and f) Comparisonof helix 3 for K137A (left) and E190A (right) from the crystal structures(e) or from MD simulations (f). In panel e, the green sphere representsa new water molecule observed in the mutants. Amino acids are coloredyellow for the mutant and gray for WT caspase-3. For panels c, d,and f, 200 frames (at 250 ps intervals) of the 50 ns simulations areshown to demonstrate movements of the amino acids.
Mentions: The enzyme activity data showedthat increasing the size of theside chain at position 55 in loop 1 correlated with a decrease inactivity (Table 2). Unfortunately, we are unableto report the structure of the F55Y variant, but we determined thestructure of F55W to 1.9 Å resolution (Table S3 of the Supporting Information). The active site groupsoverlay well with those of wild-type caspase-3with the exception of M61, which is displaced because of the largetryptophan side chain. In addition, the indole nitrogen forms a newH-bond with the carbonyl of G129, which resides on a turn betweenβ-strands 4 and 5 (Figure 8a). Whilethe electron density is good for W55 in both active sites, severalresidues in loop 1 (H56–T62) are disordered in one active site,indicating that the H-bond between the catalytic H121 and the backbonecarbonyl of T62 is disrupted (Figure 8b). Inthe interface, the six conserved water molecules are present in themutant, and the interface overlays very closely with that of wild-typecaspase-3 (Figure S6d of the Supporting Information). MD simulations of the F55W variant show that the H-bond betweenW55 and G129 is maintained throughout the simulation, resulting ina lower mobility of M61 (Figure 8c). In oneactive site, the side chain of H121 rotates toward C163, as describedabove for other mutants (Figure 8c, left panel).In the second active site, however, H121 remains H-bonded to the carbonylof T62 in loop 1; that is, it does not rotate toward C163 (Figure 8c, right panel). In both active sites, M61 remainsin close contact with the hydrophobic cluster of F128 and W55 (Figure 8C). Finally, there are no major changes in the dimerinterface (Figure S6d of the Supporting Information), and helix 3 remains stable, as shown by the lack of rotation inK137 and K138 (Figure 8d).

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