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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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Moleculardynamics simulations of active site restorative mutants.MD simulations of (a and b) F128A/V266H and (c and d) M61A/V266H.(a) Comparison of active site residues for F128A/V266H at time zero(gray) and 18 ns (yellow) demonstrates movement of H121 toward C163.(b) Two hundred frames (at 250 ps intervals) of the 50 ns simulationfor F128A/V266H demonstrate movements of the amino acids in the dimerinterface. (c) The position of helix 3 is shown at time zero (gray)and 50 ns (yellow) for M61A/V266H. (d) Two hundred frames (at 250ps intervals) of the 50 ns simulation for M61A/V266H demonstrate movementsof the amino acids in the dimer interface. (e) Comparison of activesites of M61A/V266H at time zero (gray) and 50 ns (yellow) demonstratemovement of H121 toward C163.
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fig6: Moleculardynamics simulations of active site restorative mutants.MD simulations of (a and b) F128A/V266H and (c and d) M61A/V266H.(a) Comparison of active site residues for F128A/V266H at time zero(gray) and 18 ns (yellow) demonstrates movement of H121 toward C163.(b) Two hundred frames (at 250 ps intervals) of the 50 ns simulationfor F128A/V266H demonstrate movements of the amino acids in the dimerinterface. (c) The position of helix 3 is shown at time zero (gray)and 50 ns (yellow) for M61A/V266H. (d) Two hundred frames (at 250ps intervals) of the 50 ns simulation for M61A/V266H demonstrate movementsof the amino acids in the dimer interface. (e) Comparison of activesites of M61A/V266H at time zero (gray) and 50 ns (yellow) demonstratemovement of H121 toward C163.

Mentions: Results from MD simulationsfor the double mutant (F128A/V266H)show that, as described above, H266 moves toward Y195 and remainsin this position throughout the simulation (Figure 6b). Elsewhere in the dimer interface, E124 remains insertedand close to R164, so it is not available to interact with K137′across the interface. In addition, the distance between the hydroxylgroups of Y195 and T140 fluctuates between 2.5 and 3.3 Å, sothe H-bond appears to be stable. Importantly, helix 3 is alsostable; that is, it does not rotate toward the interface as describedfor the T140G/V266H variant (Figure 5f). Likethe single mutant (F128A), M61 does not block H121 but rather remainsnear F55 and A128 (Figure 6b). For this mutant,the MD simulations suggest that the presence of F128 on β-strand4 is important for maintaining optimal flexibility in active siteloop 1. A decreased flexibility in loop 1 may correlate with the decreasein kcat for the single and double mutants.


Modifying caspase-3 activity by altering allosteric networks.

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

Moleculardynamics simulations of active site restorative mutants.MD simulations of (a and b) F128A/V266H and (c and d) M61A/V266H.(a) Comparison of active site residues for F128A/V266H at time zero(gray) and 18 ns (yellow) demonstrates movement of H121 toward C163.(b) Two hundred frames (at 250 ps intervals) of the 50 ns simulationfor F128A/V266H demonstrate movements of the amino acids in the dimerinterface. (c) The position of helix 3 is shown at time zero (gray)and 50 ns (yellow) for M61A/V266H. (d) Two hundred frames (at 250ps intervals) of the 50 ns simulation for M61A/V266H demonstrate movementsof the amino acids in the dimer interface. (e) Comparison of activesites of M61A/V266H at time zero (gray) and 50 ns (yellow) demonstratemovement of H121 toward C163.
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fig6: Moleculardynamics simulations of active site restorative mutants.MD simulations of (a and b) F128A/V266H and (c and d) M61A/V266H.(a) Comparison of active site residues for F128A/V266H at time zero(gray) and 18 ns (yellow) demonstrates movement of H121 toward C163.(b) Two hundred frames (at 250 ps intervals) of the 50 ns simulationfor F128A/V266H demonstrate movements of the amino acids in the dimerinterface. (c) The position of helix 3 is shown at time zero (gray)and 50 ns (yellow) for M61A/V266H. (d) Two hundred frames (at 250ps intervals) of the 50 ns simulation for M61A/V266H demonstrate movementsof the amino acids in the dimer interface. (e) Comparison of activesites of M61A/V266H at time zero (gray) and 50 ns (yellow) demonstratemovement of H121 toward C163.
Mentions: Results from MD simulationsfor the double mutant (F128A/V266H)show that, as described above, H266 moves toward Y195 and remainsin this position throughout the simulation (Figure 6b). Elsewhere in the dimer interface, E124 remains insertedand close to R164, so it is not available to interact with K137′across the interface. In addition, the distance between the hydroxylgroups of Y195 and T140 fluctuates between 2.5 and 3.3 Å, sothe H-bond appears to be stable. Importantly, helix 3 is alsostable; that is, it does not rotate toward the interface as describedfor the T140G/V266H variant (Figure 5f). Likethe single mutant (F128A), M61 does not block H121 but rather remainsnear F55 and A128 (Figure 6b). For this mutant,the MD simulations suggest that the presence of F128 on β-strand4 is important for maintaining optimal flexibility in active siteloop 1. A decreased flexibility in loop 1 may correlate with the decreasein kcat for the single and double mutants.

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