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Hidden alternative structures of proline isomerase essential for catalysis.

Fraser JS, Clarkson MW, Degnan SC, Erion R, Kern D, Alber T - Nature (2009)

Bottom Line: A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis.Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA).This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology/QB3, University of California, Berkeley, California 94720-3220, USA.

ABSTRACT
A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.

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The structure of the Ser99Thr mutant resembles the minor conformer of wild-type CypAa,χ1 Ringer plot (0.3σ threshold is shown as yellow line) of the Ser99Thr mutant (dashed green) and room-temperature, wild-type Ser99 CypA structure (red) show that Thr99 occupies both positions populated by the Ser99-OHγ group. The angular offset between the major peaks reflects a backbone shift. b, The 2Fo-Fc simulated-annealing omit electron density map of the Ser99Thr CypA mutant (1.0σ (dark blue) and 0.3σ (light blue)) shows apparently unique conformations for Thr99 and Phe113. The structure confirmed the prediction that rotation of Phe113 to the “out” position is coupled to rotation of the Ser99 hydroxyl to the minor rotamer. c, Phe113 and Met61 in Ser99Thr CypA (green, right) are detected exclusively in the position of the minor state of the wild-type enzyme (orange, left).
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Figure 2: The structure of the Ser99Thr mutant resembles the minor conformer of wild-type CypAa,χ1 Ringer plot (0.3σ threshold is shown as yellow line) of the Ser99Thr mutant (dashed green) and room-temperature, wild-type Ser99 CypA structure (red) show that Thr99 occupies both positions populated by the Ser99-OHγ group. The angular offset between the major peaks reflects a backbone shift. b, The 2Fo-Fc simulated-annealing omit electron density map of the Ser99Thr CypA mutant (1.0σ (dark blue) and 0.3σ (light blue)) shows apparently unique conformations for Thr99 and Phe113. The structure confirmed the prediction that rotation of Phe113 to the “out” position is coupled to rotation of the Ser99 hydroxyl to the minor rotamer. c, Phe113 and Met61 in Ser99Thr CypA (green, right) are detected exclusively in the position of the minor state of the wild-type enzyme (orange, left).

Mentions: To critically test the idea that these two conformers interconvert during turnover, we designed a mutation distant from the active site to stabilize the minor CypA substate. Ser99, a buried residue in the dynamic network located >14 Å from the catalytic Arg55, was replaced by Thr to fill the space occupied by both Ser99 rotamers. This conservative change was designed to stabilize Phe113 in the “out” position by emulating the steric clash between the minor Ser99 rotamer and the Phe113 “in” position. Crystal structures of the Ser99Thr mutant, solved at 1.6-Å and 2.3-Å resolution, indeed showed Thr99 mimicking the alternate Ser99 conformations, and Phe113 was detected only in the exposed “out” rotamer (Fig. 2a, Supplementary Fig. 4). This change in rotamer populations was buttressed by 3-bond J-coupling solution NMR experiments showing that the dominant Phe113 χ1 angle changed from +60° in wild-type CypA to −60° in the Ser99Thr mutant (Supplementary Table 2). In the Ser99Thr variant structures, Thr 99, Phe113 and Met61 occupy the minor rotamers. The positions of Leu98 and Arg55 are consistent with either of the rotamers seen in wild-type CypA. This pattern corroborates the conclusions that Ser99, Phe113, Met61 and possibly Arg55 are conformationally coupled (Fig. 2c) and that the Ser99Thr mutation severely reduces the population of the major conformation seen in wild-type CypA.


Hidden alternative structures of proline isomerase essential for catalysis.

Fraser JS, Clarkson MW, Degnan SC, Erion R, Kern D, Alber T - Nature (2009)

The structure of the Ser99Thr mutant resembles the minor conformer of wild-type CypAa,χ1 Ringer plot (0.3σ threshold is shown as yellow line) of the Ser99Thr mutant (dashed green) and room-temperature, wild-type Ser99 CypA structure (red) show that Thr99 occupies both positions populated by the Ser99-OHγ group. The angular offset between the major peaks reflects a backbone shift. b, The 2Fo-Fc simulated-annealing omit electron density map of the Ser99Thr CypA mutant (1.0σ (dark blue) and 0.3σ (light blue)) shows apparently unique conformations for Thr99 and Phe113. The structure confirmed the prediction that rotation of Phe113 to the “out” position is coupled to rotation of the Ser99 hydroxyl to the minor rotamer. c, Phe113 and Met61 in Ser99Thr CypA (green, right) are detected exclusively in the position of the minor state of the wild-type enzyme (orange, left).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2805857&req=5

Figure 2: The structure of the Ser99Thr mutant resembles the minor conformer of wild-type CypAa,χ1 Ringer plot (0.3σ threshold is shown as yellow line) of the Ser99Thr mutant (dashed green) and room-temperature, wild-type Ser99 CypA structure (red) show that Thr99 occupies both positions populated by the Ser99-OHγ group. The angular offset between the major peaks reflects a backbone shift. b, The 2Fo-Fc simulated-annealing omit electron density map of the Ser99Thr CypA mutant (1.0σ (dark blue) and 0.3σ (light blue)) shows apparently unique conformations for Thr99 and Phe113. The structure confirmed the prediction that rotation of Phe113 to the “out” position is coupled to rotation of the Ser99 hydroxyl to the minor rotamer. c, Phe113 and Met61 in Ser99Thr CypA (green, right) are detected exclusively in the position of the minor state of the wild-type enzyme (orange, left).
Mentions: To critically test the idea that these two conformers interconvert during turnover, we designed a mutation distant from the active site to stabilize the minor CypA substate. Ser99, a buried residue in the dynamic network located >14 Å from the catalytic Arg55, was replaced by Thr to fill the space occupied by both Ser99 rotamers. This conservative change was designed to stabilize Phe113 in the “out” position by emulating the steric clash between the minor Ser99 rotamer and the Phe113 “in” position. Crystal structures of the Ser99Thr mutant, solved at 1.6-Å and 2.3-Å resolution, indeed showed Thr99 mimicking the alternate Ser99 conformations, and Phe113 was detected only in the exposed “out” rotamer (Fig. 2a, Supplementary Fig. 4). This change in rotamer populations was buttressed by 3-bond J-coupling solution NMR experiments showing that the dominant Phe113 χ1 angle changed from +60° in wild-type CypA to −60° in the Ser99Thr mutant (Supplementary Table 2). In the Ser99Thr variant structures, Thr 99, Phe113 and Met61 occupy the minor rotamers. The positions of Leu98 and Arg55 are consistent with either of the rotamers seen in wild-type CypA. This pattern corroborates the conclusions that Ser99, Phe113, Met61 and possibly Arg55 are conformationally coupled (Fig. 2c) and that the Ser99Thr mutation severely reduces the population of the major conformation seen in wild-type CypA.

Bottom Line: A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis.Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA).This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology/QB3, University of California, Berkeley, California 94720-3220, USA.

ABSTRACT
A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.

Show MeSH
Related in: MedlinePlus