Limits...
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.

Show MeSH

Related in: MedlinePlus

Structures of restorativemutants. Dimer interface of (a) Y195F/V266H,(b) Y195A/V266H, (c and d) T140G/V266H, (e) F128A/V266H, and (f) M61A/V266H.For panels a–f, amino acids are colored yellow for the mutantand gray for WT caspase-3. In panel c, electron density is shown byblack mesh. In panel d, the position of Y197 is compared to that ofcaspase-7 with an allosteric inhibitor, DICA, bound (pink, PDB entry 1SHJ).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263430&req=5

fig4: Structures of restorativemutants. Dimer interface of (a) Y195F/V266H,(b) Y195A/V266H, (c and d) T140G/V266H, (e) F128A/V266H, and (f) M61A/V266H.For panels a–f, amino acids are colored yellow for the mutantand gray for WT caspase-3. In panel c, electron density is shown byblack mesh. In panel d, the position of Y197 is compared to that ofcaspase-7 with an allosteric inhibitor, DICA, bound (pink, PDB entry 1SHJ).

Mentions: We determined the crystalstructures for the five restorative mutantsas well as the single-mutant controls to greater than 2 Å resolution(Table S3 of the Supporting Information). The structures of all control mutants were very similar to thatof wild-type caspase-3, except as noted below, with rmsds of <0.3Åfor all proteins (Table S2 of the SupportingInformation); however, the structures of the double mutantsshowed features of the wild type as well as those of the V266H variant.Wild-type caspase-3 contains six conserved water molecules that H-bondto R164 and R164′ across the dimer interface (see the examplein Figure S3a of the Supporting Information). In the case of double mutant Y195F/V266H, four of the conservedcentral waters were displaced by H266 and H266′, the same asobserved in single mutant V266H. The remaining two of the six conservedwaters were replaced by cryoprotectant MPD, which H-bonds to the hydroxylof Y197 (Figure 4a). In addition, H266 is observedin two conformations for one protomer and a single orientation inthe second protomer. The side chain of H266 is rotated toward Y197(and faces R164), or it is rotated toward F195. In the active siteof caspase-3(Y195F/V266H), F128 is positioned between that of F128in the wild-type or V266H structures, and M61 is observed in multipleconformations that limit clashes with H121 (Figure S2a of the Supporting Information).


Modifying caspase-3 activity by altering allosteric networks.

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

Structures of restorativemutants. Dimer interface of (a) Y195F/V266H,(b) Y195A/V266H, (c and d) T140G/V266H, (e) F128A/V266H, and (f) M61A/V266H.For panels a–f, amino acids are colored yellow for the mutantand gray for WT caspase-3. In panel c, electron density is shown byblack mesh. In panel d, the position of Y197 is compared to that ofcaspase-7 with an allosteric inhibitor, DICA, bound (pink, PDB entry 1SHJ).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Structures of restorativemutants. Dimer interface of (a) Y195F/V266H,(b) Y195A/V266H, (c and d) T140G/V266H, (e) F128A/V266H, and (f) M61A/V266H.For panels a–f, amino acids are colored yellow for the mutantand gray for WT caspase-3. In panel c, electron density is shown byblack mesh. In panel d, the position of Y197 is compared to that ofcaspase-7 with an allosteric inhibitor, DICA, bound (pink, PDB entry 1SHJ).
Mentions: We determined the crystalstructures for the five restorative mutantsas well as the single-mutant controls to greater than 2 Å resolution(Table S3 of the Supporting Information). The structures of all control mutants were very similar to thatof wild-type caspase-3, except as noted below, with rmsds of <0.3Åfor all proteins (Table S2 of the SupportingInformation); however, the structures of the double mutantsshowed features of the wild type as well as those of the V266H variant.Wild-type caspase-3 contains six conserved water molecules that H-bondto R164 and R164′ across the dimer interface (see the examplein Figure S3a of the Supporting Information). In the case of double mutant Y195F/V266H, four of the conservedcentral waters were displaced by H266 and H266′, the same asobserved in single mutant V266H. The remaining two of the six conservedwaters were replaced by cryoprotectant MPD, which H-bonds to the hydroxylof Y197 (Figure 4a). In addition, H266 is observedin two conformations for one protomer and a single orientation inthe second protomer. The side chain of H266 is rotated toward Y197(and faces R164), or it is rotated toward F195. In the active siteof caspase-3(Y195F/V266H), F128 is positioned between that of F128in the wild-type or V266H structures, and M61 is observed in multipleconformations that limit clashes with H121 (Figure S2a of the Supporting Information).

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