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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 the positions of H121 and M61 in wild-typecaspase-3.X-ray crystal structures of wild-type caspase-3 with Ac-DEVD-CMK inhibitor(gray) (PDB entry 2J30) or Ac-LDESD-CHO inhibitor (yellow) (PDB entry 3EDQ). The structurewith a pentapeptide inhibitor demonstrates two positions for H121in both active sites. In addition, in active site 1, M61 is positionedtoward solvent and blocks rotation of H121 toward loop 1. In activesite 2, M61 retains contacts with the hydrophobic cluster of F55 andF128. Similar conformations are observed for M61 and H121 in moleculardynamics simulations of caspase-3 variants, as described in the text.
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fig9: Comparison of the positions of H121 and M61 in wild-typecaspase-3.X-ray crystal structures of wild-type caspase-3 with Ac-DEVD-CMK inhibitor(gray) (PDB entry 2J30) or Ac-LDESD-CHO inhibitor (yellow) (PDB entry 3EDQ). The structurewith a pentapeptide inhibitor demonstrates two positions for H121in both active sites. In addition, in active site 1, M61 is positionedtoward solvent and blocks rotation of H121 toward loop 1. In activesite 2, M61 retains contacts with the hydrophobic cluster of F55 andF128. Similar conformations are observed for M61 and H121 in moleculardynamics simulations of caspase-3 variants, as described in the text.

Mentions: A consistent themein the caspase-3 mutants described here is thetransient rotation of the catalytic H121 toward C163. Along with thisrotation, M61, and loop 1 in general, rotates toward active site loop4. The movement results in positioning M61 toward solvent, which preventsH121 from returning to its starting position, that is, hydrogen-bondedwith T62. We note that the conformations of H121 and of M61 shownin our MD simulations were also observed in structural studies ofcaspase-3 bound to the pentapeptide inhibitor, Ac-LDESD-CHO (Figure 9). Weber and colleagues showed crystallographicevidence of M61 rotated toward solvent,28 where M61 occupies the position of H121 if it were H-bondedto T62. Thus, H121 cannot rotate toward T62 unless M61 rotates towardthe hydrophobic cluster of F55 and F128. The movements of H121 havebeen suggested to be important for the catalytic mechanism of caspases,29 and our data suggest that the movement is coordinatedwith mobility in loop 1. The data from MD simulations presented herefor the allosteric mutants at positions 55 and 128 show a change inmobility of active site loop 1, suggesting that the loop dynamicsare critical for maximal enzyme activity. Changing the mobility ofloop 1, either increasing or decreasing mobility, results in a loweractivity. The conclusions are consistent with structural studies ofother caspases, where it is well-known that caspases are inhibitedthrough conformational changes in loop1. For example, in the zymogen of caspase-8, loop 1 is locked in theactive site cleft through interactions with the intersubunit linker.30 A similar orientation was suggested for caspase-3inhibitedby calbindin-D28K, where two helices in the EF-hand 1 region lockloop 1 in the substrate-binding cleft.31 Likewise, high mobility is suggested by the disorderof loop 1 in recently described structures of procaspase-3.32


Modifying caspase-3 activity by altering allosteric networks.

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

Comparison of the positions of H121 and M61 in wild-typecaspase-3.X-ray crystal structures of wild-type caspase-3 with Ac-DEVD-CMK inhibitor(gray) (PDB entry 2J30) or Ac-LDESD-CHO inhibitor (yellow) (PDB entry 3EDQ). The structurewith a pentapeptide inhibitor demonstrates two positions for H121in both active sites. In addition, in active site 1, M61 is positionedtoward solvent and blocks rotation of H121 toward loop 1. In activesite 2, M61 retains contacts with the hydrophobic cluster of F55 andF128. Similar conformations are observed for M61 and H121 in moleculardynamics simulations of caspase-3 variants, as described in the text.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Comparison of the positions of H121 and M61 in wild-typecaspase-3.X-ray crystal structures of wild-type caspase-3 with Ac-DEVD-CMK inhibitor(gray) (PDB entry 2J30) or Ac-LDESD-CHO inhibitor (yellow) (PDB entry 3EDQ). The structurewith a pentapeptide inhibitor demonstrates two positions for H121in both active sites. In addition, in active site 1, M61 is positionedtoward solvent and blocks rotation of H121 toward loop 1. In activesite 2, M61 retains contacts with the hydrophobic cluster of F55 andF128. Similar conformations are observed for M61 and H121 in moleculardynamics simulations of caspase-3 variants, as described in the text.
Mentions: A consistent themein the caspase-3 mutants described here is thetransient rotation of the catalytic H121 toward C163. Along with thisrotation, M61, and loop 1 in general, rotates toward active site loop4. The movement results in positioning M61 toward solvent, which preventsH121 from returning to its starting position, that is, hydrogen-bondedwith T62. We note that the conformations of H121 and of M61 shownin our MD simulations were also observed in structural studies ofcaspase-3 bound to the pentapeptide inhibitor, Ac-LDESD-CHO (Figure 9). Weber and colleagues showed crystallographicevidence of M61 rotated toward solvent,28 where M61 occupies the position of H121 if it were H-bondedto T62. Thus, H121 cannot rotate toward T62 unless M61 rotates towardthe hydrophobic cluster of F55 and F128. The movements of H121 havebeen suggested to be important for the catalytic mechanism of caspases,29 and our data suggest that the movement is coordinatedwith mobility in loop 1. The data from MD simulations presented herefor the allosteric mutants at positions 55 and 128 show a change inmobility of active site loop 1, suggesting that the loop dynamicsare critical for maximal enzyme activity. Changing the mobility ofloop 1, either increasing or decreasing mobility, results in a loweractivity. The conclusions are consistent with structural studies ofother caspases, where it is well-known that caspases are inhibitedthrough conformational changes in loop1. For example, in the zymogen of caspase-8, loop 1 is locked in theactive site cleft through interactions with the intersubunit linker.30 A similar orientation was suggested for caspase-3inhibitedby calbindin-D28K, where two helices in the EF-hand 1 region lockloop 1 in the substrate-binding cleft.31 Likewise, high mobility is suggested by the disorderof loop 1 in recently described structures of procaspase-3.32

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