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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 Ser99Thr mutation shifts the equilibrium toward the minor wild-type conformation and slows motions in the dynamic network in free CypAa, Significant 1H-15N chemical-shift differences between Ser99Thr and wild-type CypA (red) propagate through group I residues (Arg55, Phe113, and Ser99 shown as black sticks). b, Linear amide chemical shift changes (arrows) between wild-type (black), Lys82Ala (red) and Ser99Thr (blue) CypA reflect the inversion of the major/minor equilibrium due to the Ser99Thr mutation. c, Residues undergoing slow (red) or fast (blue) motions on the NMR time scale in Ser99Thr (right) coincide with previously identified group I (red) and group II (blue) residues in wild-type (left) CypA (amides in grey are prolines or overlapped peaks). d, Temperature dependence of CPMG 15N NMR relaxation data for group I (left) and group II (right) in Ser99Thr CypA reveal that the mutation impedes group I conformational dynamics (REX ~ k1 and REX increases with temperature). In contrast, group II residues are unaffected by the mutation and display the opposite temperature dependence characteristic of fast motions on the NMR time scale. Dispersion curves were normalized to R20 at 30 °C.
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Figure 3: The Ser99Thr mutation shifts the equilibrium toward the minor wild-type conformation and slows motions in the dynamic network in free CypAa, Significant 1H-15N chemical-shift differences between Ser99Thr and wild-type CypA (red) propagate through group I residues (Arg55, Phe113, and Ser99 shown as black sticks). b, Linear amide chemical shift changes (arrows) between wild-type (black), Lys82Ala (red) and Ser99Thr (blue) CypA reflect the inversion of the major/minor equilibrium due to the Ser99Thr mutation. c, Residues undergoing slow (red) or fast (blue) motions on the NMR time scale in Ser99Thr (right) coincide with previously identified group I (red) and group II (blue) residues in wild-type (left) CypA (amides in grey are prolines or overlapped peaks). d, Temperature dependence of CPMG 15N NMR relaxation data for group I (left) and group II (right) in Ser99Thr CypA reveal that the mutation impedes group I conformational dynamics (REX ~ k1 and REX increases with temperature). In contrast, group II residues are unaffected by the mutation and display the opposite temperature dependence characteristic of fast motions on the NMR time scale. Dispersion curves were normalized to R20 at 30 °C.

Mentions: Connecting this interpretation of the crystal structures to the solution behaviour of the enzyme, NMR detected amide chemical-shift differences between wild-type and Ser99Thr CypA in most active-site and core residues of the dynamic network (Fig. 3a,b). Although it is tempting to speculate that the Ser99Thr mutation “traps” the minor state of wild-type CypA based on our crystallographic data (Fig. 2), NMR relaxation-dispersion analysis18 of the mutant enzyme revealed conformational exchange in both regions that showed collective motions in the wild-type enzyme (Fig. 3c). These regions include residues coupled to the active site (group I) and residues in the 65-85 loops (group II)4. In addition, for most of the group I residues, the peaks in Ser99Thr CypA shift relative to wild-type in the same direction as peaks in the previously characterized Lys82Ala variant, which displays a small increase in the population of the minor state4. The Ser99Thr mutation, however, causes much larger shifts (Fig. 3b, Supplementary Figs. 5,6), indicating that the populations are inverted. Together with our crystallographic and NMR J-coupling data, these results show that the Ser99Thr mutation shifts the structural equilibrium strongly towards a conformation that recapitulates key features of the previously undefined minor state of 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 Ser99Thr mutation shifts the equilibrium toward the minor wild-type conformation and slows motions in the dynamic network in free CypAa, Significant 1H-15N chemical-shift differences between Ser99Thr and wild-type CypA (red) propagate through group I residues (Arg55, Phe113, and Ser99 shown as black sticks). b, Linear amide chemical shift changes (arrows) between wild-type (black), Lys82Ala (red) and Ser99Thr (blue) CypA reflect the inversion of the major/minor equilibrium due to the Ser99Thr mutation. c, Residues undergoing slow (red) or fast (blue) motions on the NMR time scale in Ser99Thr (right) coincide with previously identified group I (red) and group II (blue) residues in wild-type (left) CypA (amides in grey are prolines or overlapped peaks). d, Temperature dependence of CPMG 15N NMR relaxation data for group I (left) and group II (right) in Ser99Thr CypA reveal that the mutation impedes group I conformational dynamics (REX ~ k1 and REX increases with temperature). In contrast, group II residues are unaffected by the mutation and display the opposite temperature dependence characteristic of fast motions on the NMR time scale. Dispersion curves were normalized to R20 at 30 °C.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: The Ser99Thr mutation shifts the equilibrium toward the minor wild-type conformation and slows motions in the dynamic network in free CypAa, Significant 1H-15N chemical-shift differences between Ser99Thr and wild-type CypA (red) propagate through group I residues (Arg55, Phe113, and Ser99 shown as black sticks). b, Linear amide chemical shift changes (arrows) between wild-type (black), Lys82Ala (red) and Ser99Thr (blue) CypA reflect the inversion of the major/minor equilibrium due to the Ser99Thr mutation. c, Residues undergoing slow (red) or fast (blue) motions on the NMR time scale in Ser99Thr (right) coincide with previously identified group I (red) and group II (blue) residues in wild-type (left) CypA (amides in grey are prolines or overlapped peaks). d, Temperature dependence of CPMG 15N NMR relaxation data for group I (left) and group II (right) in Ser99Thr CypA reveal that the mutation impedes group I conformational dynamics (REX ~ k1 and REX increases with temperature). In contrast, group II residues are unaffected by the mutation and display the opposite temperature dependence characteristic of fast motions on the NMR time scale. Dispersion curves were normalized to R20 at 30 °C.
Mentions: Connecting this interpretation of the crystal structures to the solution behaviour of the enzyme, NMR detected amide chemical-shift differences between wild-type and Ser99Thr CypA in most active-site and core residues of the dynamic network (Fig. 3a,b). Although it is tempting to speculate that the Ser99Thr mutation “traps” the minor state of wild-type CypA based on our crystallographic data (Fig. 2), NMR relaxation-dispersion analysis18 of the mutant enzyme revealed conformational exchange in both regions that showed collective motions in the wild-type enzyme (Fig. 3c). These regions include residues coupled to the active site (group I) and residues in the 65-85 loops (group II)4. In addition, for most of the group I residues, the peaks in Ser99Thr CypA shift relative to wild-type in the same direction as peaks in the previously characterized Lys82Ala variant, which displays a small increase in the population of the minor state4. The Ser99Thr mutation, however, causes much larger shifts (Fig. 3b, Supplementary Figs. 5,6), indicating that the populations are inverted. Together with our crystallographic and NMR J-coupling data, these results show that the Ser99Thr mutation shifts the structural equilibrium strongly towards a conformation that recapitulates key features of the previously undefined minor state of 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