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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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Room-temperature X-ray crystallography and Ringer analysis detect conformational substates in CypAa, Local maxima above the 0.3σ threshold (yellow line) in Ringer plots13 reveal alternate side-chain conformations in room-temperature (red line) but not cryogenic (blue line) electron density for Ser99, Leu98, Met61 and Arg55. b, Electron-density maps calculated using room-temperature X-ray data define the alternate conformers of Leu98, Ser99 and Phe113. 2Fo-Fc electron density (blue mesh; 1σ); positive (green) and negative (red) Fo-Fc difference density (3σ). c, 2Fo-Fc composite simulated-annealing omit electron density maps (1.0σ (dark blue) and 0.3σ (light blue)) show a unique conformation for Phe113 in the 1.2-Å-resolution cryogenic structure (blue) and distinct major (red) and minor (orange) conformers in the 1.39-Å-resolution room-temperature structure. Electron density around the main chain and the surrounding residues was omitted for clarity. d, Steric collisions across the network of major (red) and minor (orange) conformers of Arg55, Met61, Phe113 and Ser99 explain how side-chain motions link the active site to remote buried residues.
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Figure 1: Room-temperature X-ray crystallography and Ringer analysis detect conformational substates in CypAa, Local maxima above the 0.3σ threshold (yellow line) in Ringer plots13 reveal alternate side-chain conformations in room-temperature (red line) but not cryogenic (blue line) electron density for Ser99, Leu98, Met61 and Arg55. b, Electron-density maps calculated using room-temperature X-ray data define the alternate conformers of Leu98, Ser99 and Phe113. 2Fo-Fc electron density (blue mesh; 1σ); positive (green) and negative (red) Fo-Fc difference density (3σ). c, 2Fo-Fc composite simulated-annealing omit electron density maps (1.0σ (dark blue) and 0.3σ (light blue)) show a unique conformation for Phe113 in the 1.2-Å-resolution cryogenic structure (blue) and distinct major (red) and minor (orange) conformers in the 1.39-Å-resolution room-temperature structure. Electron density around the main chain and the surrounding residues was omitted for clarity. d, Steric collisions across the network of major (red) and minor (orange) conformers of Arg55, Met61, Phe113 and Ser99 explain how side-chain motions link the active site to remote buried residues.

Mentions: To address this discrepancy between the X-ray and NMR analyses, we collected 1.39-Å diffraction data at ambient temperature to explore the possibility that the low-temperature data collection might alter the conformational distribution in the crystal15,16. Ringer plots indeed uncovered 0.3-1 σ features for alternate rotamers of several residues including Leu98 and Ser99, in addition to the side-chain heterogeneity observed at cryogenic temperature (Fig. 1a). These results emphasize that crystal freezing can alter conformational distributions.


Hidden alternative structures of proline isomerase essential for catalysis.

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

Room-temperature X-ray crystallography and Ringer analysis detect conformational substates in CypAa, Local maxima above the 0.3σ threshold (yellow line) in Ringer plots13 reveal alternate side-chain conformations in room-temperature (red line) but not cryogenic (blue line) electron density for Ser99, Leu98, Met61 and Arg55. b, Electron-density maps calculated using room-temperature X-ray data define the alternate conformers of Leu98, Ser99 and Phe113. 2Fo-Fc electron density (blue mesh; 1σ); positive (green) and negative (red) Fo-Fc difference density (3σ). c, 2Fo-Fc composite simulated-annealing omit electron density maps (1.0σ (dark blue) and 0.3σ (light blue)) show a unique conformation for Phe113 in the 1.2-Å-resolution cryogenic structure (blue) and distinct major (red) and minor (orange) conformers in the 1.39-Å-resolution room-temperature structure. Electron density around the main chain and the surrounding residues was omitted for clarity. d, Steric collisions across the network of major (red) and minor (orange) conformers of Arg55, Met61, Phe113 and Ser99 explain how side-chain motions link the active site to remote buried residues.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2805857&req=5

Figure 1: Room-temperature X-ray crystallography and Ringer analysis detect conformational substates in CypAa, Local maxima above the 0.3σ threshold (yellow line) in Ringer plots13 reveal alternate side-chain conformations in room-temperature (red line) but not cryogenic (blue line) electron density for Ser99, Leu98, Met61 and Arg55. b, Electron-density maps calculated using room-temperature X-ray data define the alternate conformers of Leu98, Ser99 and Phe113. 2Fo-Fc electron density (blue mesh; 1σ); positive (green) and negative (red) Fo-Fc difference density (3σ). c, 2Fo-Fc composite simulated-annealing omit electron density maps (1.0σ (dark blue) and 0.3σ (light blue)) show a unique conformation for Phe113 in the 1.2-Å-resolution cryogenic structure (blue) and distinct major (red) and minor (orange) conformers in the 1.39-Å-resolution room-temperature structure. Electron density around the main chain and the surrounding residues was omitted for clarity. d, Steric collisions across the network of major (red) and minor (orange) conformers of Arg55, Met61, Phe113 and Ser99 explain how side-chain motions link the active site to remote buried residues.
Mentions: To address this discrepancy between the X-ray and NMR analyses, we collected 1.39-Å diffraction data at ambient temperature to explore the possibility that the low-temperature data collection might alter the conformational distribution in the crystal15,16. Ringer plots indeed uncovered 0.3-1 σ features for alternate rotamers of several residues including Leu98 and Ser99, in addition to the side-chain heterogeneity observed at cryogenic temperature (Fig. 1a). These results emphasize that crystal freezing can alter conformational distributions.

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