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Hidden alternative structures of proline isomerase essential for catalysis

Nature volume 462, pages 669–673 (2009)Cite this article

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 reactions1, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations1,2. 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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Figure 1: Room-temperature X-ray crystallography and Ringer analysis detect conformational substates in CYPA.
Figure 2: The structure of the Ser99Thr mutant resembles the minor conformer of wild-type CYPA.
Figure 3: The Ser99Thr mutation shifts the equilibrium towards the minor wild-type conformation and slows motions in the dynamic network in free CYPA.
Figure 4: Impeded motions in the dynamic network severely reduce the catalytic power of a chemically competent enzyme.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the PDB under accession codes 3K0M, 3K0N, 3K0O, 3K0P, 3K0Q and 3K0R.

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Acknowledgements

We thank S. Marqusee and B. Krantz for discussions; S. Classen, G. Meigs, J. Holton, A. Samelson, N. Echols, P. Afonine, and the Phenix team for technical support; J. Tainer for access to Rigaku free-mounting device at ALS Beamline 12.3.1; J. Pelton and D. Wemmer for providing essential help and access to NMR facilities. J.S.F. was supported by US NSF and Canadian NSERC fellowships. This work was funded by the US National Institutes of Health (to T.A.) and the US National Institutes of Health, the US Department of Energy Office of Basic Energy Sciences, and the Howard Hughes Medical Institute (to D.K.).

Author Contributions J.S.F., S.C.D. and R.E. performed the X-ray experiments, M.W.C. performed the NMR experiments, and M.W.C. and J.S.F. performed the activity and binding assays. J.S.F., M.W.C., D.K. and T.A. analysed data and wrote the paper. All authors contributed to data interpretation and commented on the manuscript.

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Authors and Affiliations

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

    James S. Fraser, Sheena C. Degnan, Renske Erion & Tom Alber

  2. Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA,

    Michael W. Clarkson & Dorothee Kern

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  1. James S. Fraser
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Corresponding authors

Correspondence to Dorothee Kern or Tom Alber.

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Fraser, J., Clarkson, M., Degnan, S. et al. Hidden alternative structures of proline isomerase essential for catalysis . Nature 462, 669–673 (2009). https://doi.org/10.1038/nature08615

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