Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Hidden alternative structures of proline isomerase essential for catalysis


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

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.


  1. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007)

    CAS  ADS  Article  Google Scholar 

  2. Mittermaier, A. & Kay, L. E. Review—new tools provide new insights in NMR studies of protein dynamics. Science 312, 224–228 (2006)

    CAS  ADS  Article  Google Scholar 

  3. Boehr, D. D., McElheny, D., Dyson, H. J. & Wright, P. E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313, 1638–1642 (2006)

    CAS  ADS  Article  Google Scholar 

  4. Eisenmesser, E. Z. et al. Intrinsic dynamics of an enzyme underlies catalysis. Nature 438, 117–121 (2005)

    CAS  ADS  Article  Google Scholar 

  5. Hammes-Schiffer, S. & Benkovic, S. J. Relating protein motion to catalysis. Annu. Rev. Biochem. 75, 519–541 (2006)

    CAS  Article  Google Scholar 

  6. Schramm, V. L. & Shi, W. Atomic motion in enzymatic reaction coordinates. Curr. Opin. Struct. Biol. 11, 657–665 (2001)

    CAS  Article  Google Scholar 

  7. Agarwal, P. K. Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: insights from computational and theoretical studies. Proteins 56, 449–463 (2004)

    CAS  Article  Google Scholar 

  8. Hamelberg, D. & McCammon, A. Mechanistic insight into the role of transition-state stabilization in cyclophilin A. J. Am. Chem. Soc. 131, 147–152 (2009)

    CAS  Article  Google Scholar 

  9. Li, G. H. & Cui, Q. What is so special about Arg 55 in the catalysis of cyclophilin A? Insights from hybrid QM/MM simulations. J. Am. Chem. Soc. 125, 15028–15038 (2003)

    CAS  Article  Google Scholar 

  10. Trzesniak, D. & Van Gunsteren, W. F. Catalytic mechanism of cyclophilin as observed in molecular dynamics simulations: pathway prediction and reconciliation of X-ray crystallographic and NMR solution data. Protein Sci. 15, 2544–2551 (2006)

    CAS  Article  Google Scholar 

  11. Howard, B. R., Vajdos, F. F., Li, S., Sundquist, W. I. & Hill, C. P. Structural insights into the catalytic mechanism of cyclophilin A. Nature Struct. Biol. 10, 475–481 (2003)

    CAS  Article  Google Scholar 

  12. Ke, H. M. & Huai, Q. Crystal structures of cyclophilin and its partners. Front. Biosci. 9, 2285–2296 (2004)

    CAS  Article  Google Scholar 

  13. Lang, P. T. et al. Automated electron-density sampling reveals widespread conformational polymorphism in proteins. Protein Sci. (submitted)

  14. Eisenmesser, E. Z., Bosco, D. A., Akke, M. & Kern, D. Enzyme dynamics during catalysis. Science 295, 1520–1523 (2002)

    CAS  ADS  Article  Google Scholar 

  15. Halle, B. Biomolecular cryocrystallography: structural changes during flash-cooling. Proc. Natl Acad. Sci. USA 101, 4793–4798 (2004)

    CAS  ADS  Article  Google Scholar 

  16. Rasmussen, B. F., Stock, A. M., Ringe, D. & Petsko, G. A. Crystalline ribonuclease A loses function below the dynamical transition at 220 K. Nature 357, 423–424 (1992)

    CAS  ADS  Article  Google Scholar 

  17. Loria, J. P., Rance, M. & Palmer, A. G. A TROSY CPMG sequence for characterizing chemical exchange in large proteins. J. Biomol. NMR 15, 151–155 (1999)

    CAS  Article  Google Scholar 

  18. Millet, O., Loria, J. P., Kroenke, C. D., Pons, M. & Palmer, A. G. The static magnetic field dependence of chemical exchange linebroadening defines the NMR chemical shift time scale. J. Am. Chem. Soc. 122, 2867–2877 (2000)

    CAS  Article  Google Scholar 

  19. Kofron, J. L., Kuzmic, P., Kishore, V., Colonbonilla, E. & Rich, D. H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry 30, 6127–6134 (1991)

    CAS  Article  Google Scholar 

  20. Farrow, N. A., Zhang, O. W., Forman-Kay, J. D. & Kay, L. E. A Heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium. J. Biomol. NMR 4, 727–734 (1994)

    CAS  Article  Google Scholar 

  21. Zydowsky, L. D. et al. Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporine A binding and calcineurin inhibition. Protein Sci. 1, 1092–1099 (1992)

    CAS  Article  Google Scholar 

  22. Frauenfelder, H. et al. Thermal expansion of a protein. Biochemistry 26, 254–261 (1987)

    CAS  Article  Google Scholar 

  23. Frauenfelder, H., Petsko, G. A. & Tsernoglou, D. Temperature-dependent X-ray diffraction as a probe of protein structural dynamics. Nature 280, 558–563 (1979)

    CAS  ADS  Article  Google Scholar 

  24. Tilton, R. F., Dewan, J. C. & Petsko, G. A. Effects of temperature on protein structure and dynamics: X-ray crystallographic studies of the protein ribonuclease-A at nine different temperatures from 98 to 320K. Biochemistry 31, 2469–2481 (1992)

    CAS  Article  Google Scholar 

  25. Beach, H., Cole, R., Gill, M. L. & Loria, J. P. Conservation of μs-ms enzyme motions in the apo- and substrate-mimicked state. J. Am. Chem. Soc. 127, 9167–9176 (2005)

    CAS  Article  Google Scholar 

  26. Tokuriki, N. & Tawfik, D. S. Protein dynamism and evolvability. Science 324, 203–207 (2009)

    CAS  ADS  Article  Google Scholar 

  27. Lee, G. M. & Craik, C. S. Trapping moving targets with small molecules. Science 324, 213–215 (2009)

    CAS  ADS  Article  Google Scholar 

  28. Kiefersauer, R. et al. A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes. J. Appl. Crystallogr. 33, 1223–1230 (2000)

    CAS  Article  Google Scholar 

  29. Davis, D. G., Perlman, M. E. & London, R. E. Direct measurements of the dissociation-rate constant for inhibitor-enzyme complexes via the T 1ρ and T 2(CPMG) methods. J. Magn. Reson. Ser. B 104, 266–275 (1994)

    CAS  Article  Google Scholar 

  30. Hu, J. S., Grzesiek, S. & Bax, A. Two-dimensional NMR methods for determining χ1 angles of aromatic residues in proteins from three-bond JC'Cγ and JNCγ couplings. J. Am. Chem. Soc. 119, 1803–1804 (1997)

    CAS  Article  Google Scholar 

  31. Southworth-Davies, R. J., Medina, M. A., Carmichael, I. & Garman, E. F. Observation of decreased radiation damage at higher dose rates in room temperature protein crystallography. Structure 15, 1531–1541 (2007)

    CAS  Article  Google Scholar 

  32. Otwinowski, Z. & Minor, W. in Macromolecular Crystallography Part A 307–326 (Methods in Enzymology, Vol. 276, Academic, 1997)

    Book  Google Scholar 

  33. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  34. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  35. Holton, J. & Alber, T. Automated protein crystal structure determination using ELVES. Proc. Natl Acad. Sci. USA 101, 1537–1542 (2004)

    CAS  ADS  Article  Google Scholar 

  36. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    CAS  Article  Google Scholar 

  37. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  38. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    ADS  Article  Google Scholar 

  39. Laskowski, R. A., Macarthur, M. W., Moss, D. S. & Thornton, J. M. Procheck—a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)

    CAS  Article  Google Scholar 

  40. Delano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, Palo Alto, 2008); 〈

  41. Theobald, D. L. & Wuttke, D. S. THESEUS: maximum likelihood superpositioning and analysis of macromolecular structures. Bioinformatics 22, 2171–2172 (2006)

    CAS  Article  Google Scholar 

  42. Mulder, F. A. A., Mittermaier, A., Hon, B., Dahlquist, F. W. & Kay, L. E. Studying excited states of proteins by NMR spectroscopy. Nature Struct. Biol. 8, 932–935 (2001)

    CAS  Article  Google Scholar 

  43. Delaglio, F. et al. NMRPipe—a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    CAS  Article  Google Scholar 

  44. Johnson, B. A. & Blevins, R. A. NMR View—a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994)

    CAS  Article  Google Scholar 

  45. Jeener, J., Meier, B. H., Bachmann, P. & Ernst, R. R. Investigation of exchange processes by 2-dimensional NMR-spectroscopy. J. Chem. Phys. 71, 4546–4553 (1979)

    CAS  ADS  Article  Google Scholar 

Download references


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.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Dorothee Kern or Tom Alber.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 with Legends and Supplementary Tables 1-4. (PDF 7505 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fraser, J., Clarkson, M., Degnan, S. et al. Hidden alternative structures of proline isomerase essential for catalysis . Nature 462, 669–673 (2009).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing