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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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Molecular dynamics simulations of interface restorativemutants.MD simulations of (a) Y195F/V266H, (b) wild type, (c) Y195A/V266H,and (d–f) T140G/V266H. Panels a–d show 200 frames (at250 ps intervals) of the 50 ns simulation to demonstrate movementsof the amino acids in the dimer interface. (e) Active site residuesfor T140G/V266H at time zero (gray) and 29 ns (yellow). (f) Positionof helix 3 at time zero (gray) and 24 ns (yellow).
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fig5: Molecular dynamics simulations of interface restorativemutants.MD simulations of (a) Y195F/V266H, (b) wild type, (c) Y195A/V266H,and (d–f) T140G/V266H. Panels a–d show 200 frames (at250 ps intervals) of the 50 ns simulation to demonstrate movementsof the amino acids in the dimer interface. (e) Active site residuesfor T140G/V266H at time zero (gray) and 29 ns (yellow). (f) Positionof helix 3 at time zero (gray) and 24 ns (yellow).

Mentions: We performed molecular dynamics simulations foreach of the mutants for a total time of 50 ns. The results for caspase-3(Y195F/V266H)show that H266 rotates toward F195 (and H266′ rotates towardF195′) within ∼15 ns, and the histidines remain in thatposition for the duration of the simulation. Both E124 and E124′remain inserted into the interface and interact with R164, while K137is very dynamic and is positioned toward solvent throughout most ofthe simulation. Interestingly, F195 is significantly more mobile thanY195. With H266 rotated toward F195, the side chain of F195 also rotatestoward β-strand 6, which is not observed for Y195 in wild-typecaspase-3 (compare panels a and b of Figure 5). In the active site of caspase-3(Y195F/V266H), H121 rotates towardC163 within the first ∼15 ns and remains in that position forthe remainder of the simulation. Following the rotation of H121, M61moves behind H121 (toward solvent) and prevents it from rotating backtoward active site loop 1 (see the example in Figure 5e). These movements are observed in one of the two activesites in wild-type caspase-3, where the distance between T62 and H121fluctuates between ∼6 and ∼9 Å. In the second activesite of wild-type caspase-3, the distance isconstant at ∼3 Å, demonstrating that the hydrogen bondbetween T62 and H121 is maintainedduring the time course of the simulation. A comparison of the distancesin wild-type caspase-3 and the M61A/V266H double mutant (as an example)is shown in Figure S7 of the Supporting Information (panels a and b).


Modifying caspase-3 activity by altering allosteric networks.

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

Molecular dynamics simulations of interface restorativemutants.MD simulations of (a) Y195F/V266H, (b) wild type, (c) Y195A/V266H,and (d–f) T140G/V266H. Panels a–d show 200 frames (at250 ps intervals) of the 50 ns simulation to demonstrate movementsof the amino acids in the dimer interface. (e) Active site residuesfor T140G/V266H at time zero (gray) and 29 ns (yellow). (f) Positionof helix 3 at time zero (gray) and 24 ns (yellow).
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fig5: Molecular dynamics simulations of interface restorativemutants.MD simulations of (a) Y195F/V266H, (b) wild type, (c) Y195A/V266H,and (d–f) T140G/V266H. Panels a–d show 200 frames (at250 ps intervals) of the 50 ns simulation to demonstrate movementsof the amino acids in the dimer interface. (e) Active site residuesfor T140G/V266H at time zero (gray) and 29 ns (yellow). (f) Positionof helix 3 at time zero (gray) and 24 ns (yellow).
Mentions: We performed molecular dynamics simulations foreach of the mutants for a total time of 50 ns. The results for caspase-3(Y195F/V266H)show that H266 rotates toward F195 (and H266′ rotates towardF195′) within ∼15 ns, and the histidines remain in thatposition for the duration of the simulation. Both E124 and E124′remain inserted into the interface and interact with R164, while K137is very dynamic and is positioned toward solvent throughout most ofthe simulation. Interestingly, F195 is significantly more mobile thanY195. With H266 rotated toward F195, the side chain of F195 also rotatestoward β-strand 6, which is not observed for Y195 in wild-typecaspase-3 (compare panels a and b of Figure 5). In the active site of caspase-3(Y195F/V266H), H121 rotates towardC163 within the first ∼15 ns and remains in that position forthe remainder of the simulation. Following the rotation of H121, M61moves behind H121 (toward solvent) and prevents it from rotating backtoward active site loop 1 (see the example in Figure 5e). These movements are observed in one of the two activesites in wild-type caspase-3, where the distance between T62 and H121fluctuates between ∼6 and ∼9 Å. In the second activesite of wild-type caspase-3, the distance isconstant at ∼3 Å, demonstrating that the hydrogen bondbetween T62 and H121 is maintainedduring the time course of the simulation. A comparison of the distancesin wild-type caspase-3 and the M61A/V266H double mutant (as an example)is shown in Figure S7 of the Supporting Information (panels a and b).

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