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Intrinsic dynamics of an enzyme underlies catalysis

Abstract

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis1,2,3,4,5. The concept of conformational substates has been described6; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cistrans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously3,7,8,9,10, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.

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Figure 1: Protein dynamics necessary for catalysis is an intrinsic property of the enzyme.
Figure 2: Identification of residues that build a common dynamic network in CypA.
Figure 3: Probing correlated motions of CypA by point mutations.

References

  1. 1

    Jencks, W. P. Catalysis in Chemistry and Enzymology (Dover, New York, 1987)

    Google Scholar 

  2. 2

    Hammes, G. Multiple conformational changes in enzyme catalysis. Biochemistry 41, 8221–8228 (2002)

    CAS  Article  Google Scholar 

  3. 3

    Benkovic, S. J. & Hammes-Schiffer, S. A perspective on enzyme catalysis. Science 301, 1196–1202 (2003)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Garcia-Viloca, M., Gao, J., Karplus, M. & Truhlar, D. G. How enzymes work: analysis by modern rate theory and computer simulations. Science 303, 186–195 (2004)

    ADS  CAS  Article  Google Scholar 

  5. 5

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

    ADS  CAS  Article  Google Scholar 

  6. 6

    Austin, R. H. et al. Dynamics of ligand binding to myoglobin. Biochemistry 14, 5355–5373 (1975)

    CAS  Article  Google Scholar 

  7. 7

    Berendsen, H. J. C. & Hayward, S. Collective protein dynamics in relation to function. Curr. Opin. Struct. Biol. 10, 165–169 (2000)

    CAS  Article  Google Scholar 

  8. 8

    Rod, T. H., Radkiewicz, J. L. & Brooks, C. L. Correlated motion and the effect of distal mutations in dihydrofolate reductase. Proc. Natl Acad. Sci. USA 100, 6980–6985 (2003)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Suel, G. M., Lockless, S. W., Wall, M. A. & Ranganathan, R. Evolutionarily conserved networks of residues mediate allosteric communication in proteins. Nature Struct. Biol. 10, 59–69 (2003)

    Article  Google Scholar 

  10. 10

    Agarwal, P. K., Geist, A. & Gorin, A. Protein dynamics and enzymatic catalysis: Investigating the peptidyl-prolyl cis-trans isomerization activity of cyclophilin A. Biochemistry 43, 10605–10618 (2004)

    CAS  Article  Google Scholar 

  11. 11

    Schmid, F. X. Prolyl isomerases. Adv. Protein Chem. 59, 243–282 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Goff, S. P. Genetic control of retrovirus susceptibility in mammalian cells. Annu. Rev. Genet. 38, 61–85 (2004)

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    Palmer, A. G. NMR characterization of the dynamics of biomacromolecules. Chem. Rev. 104, 3623–3640 (2004)

    CAS  Article  Google Scholar 

  15. 15

    Kern, D., Kern, G., Scherer, G., Fischer, G. & Drakenberg, T. Kinetic analysis of cyclophilin-catalyzed prolyl cis/trans isomerization by dynamic NMR spectroscopy. Biochemistry 34, 13594–13602 (1995)

    CAS  Article  Google Scholar 

  16. 16

    Zhao, Y. & Ke, H. Crystal structure implies that cyclophilin predominantly catalyzes the trans to cis isomerization. Biochemistry 35, 7356–7361 (1996)

    CAS  Article  Google Scholar 

  17. 17

    Fischer, G., Bang, H. & Mech, C. Determination of enzymatic catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides. Biomed. Biochim. Acta 43, 1101–1111 (1984)

    CAS  PubMed  Google Scholar 

  18. 18

    Ottiger, M., Zerbe, O., Guntert, P. & Wuthrich, K. The NMR solution conformation of unligated human cyclophilin A. J. Mol. Biol. 272, 64–81 (1997)

    CAS  Article  Google Scholar 

  19. 19

    Skrynnikov, N. R., Dahlquist, F. W. & Kay, L. E. Reconstructing NMR spectra of ‘invisible’ excited protein states using HSQC and HMQC experiments. J. Am. Chem. Soc. 124, 12352–12360 (2002)

    CAS  Article  Google Scholar 

  20. 20

    Wishart, D. S., Sykes, B. D. & Richards, F. M. Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J. Mol. Biol. 222, 311–333 (1991)

    CAS  Article  Google Scholar 

  21. 21

    Rozovsky, S., Jogl, G., Tong, L. & McKermott, A. E. Solution-state NMR investigations of triosephosphate isomerase active site loop motion: ligand release in relation to active site loop dynamics. J. Mol. Biol. 310, 271–280 (2001)

    CAS  Article  Google Scholar 

  22. 22

    Williams, J. C. & McDermott, A. E. Dynamics of the flexible loop of triosephosphate isomerase: the loop motion is not ligand gated. Biochemistry 34, 8309–8319 (1995)

    CAS  Article  Google Scholar 

  23. 23

    Schnell, J. R., Dyson, H. J. & Wright, P. E. Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu. Rev. Biophys. Biomol. Struct. 33, 119–140 (2004)

    CAS  Article  Google Scholar 

  24. 24

    McElheny, D., Schnell, J. R., Lansing, J. C., Dyson, H. J. & Wright, P. E. Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc. Natl Acad. Sci. USA 102, 5032–5037 (2005)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Ishima, R., Freedberg, D. I., Wang, Y. X., Louis, J. M. & Torchia, D. A. Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function. Struct. Fold. Des. 7, 1047–1055 (1999)

    CAS  Article  Google Scholar 

  26. 26

    Cole, R. & Loria, J. P. Evidence for flexibility in the function of ribonuclease A. Biochemistry 41, 6072–6081 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Volkman, B. F., Lipson, D., Wemmer, D. E. & Kern, D. Two-state allosteric behaviour in a single-domain signalling protein. Science 291, 2429–2433 (2001)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Rosen, M. K. et al. Selective methyl group protonation of perdeuterated proteins. J. Mol. Biol. 263, 627–636 (1996)

    CAS  Article  Google Scholar 

  29. 29

    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 

  30. 30

    Carver, J. P. & Richards, R. E. A general two-site solution for the chemical exchange produced dependence of T2 upon the Carr-Purcell pulse separation. J. Magn. Reson. 6, 89–105 (1972)

    ADS  CAS  Google Scholar 

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Acknowledgements

We thank K. H. Wildman for discussions. This work was supported by NIH grants to D.K., by a grant from the Canadian Institutes of Health Research to L.E.K., and by a grant from the Swedish Research Council to M.W.W. Part of the NMR studies was performed at the NHMFL at Florida with support from the NSF.

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Correspondence to Dorothee Kern.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Concentration dependence of relaxation dispersion data for CypA. (PDF 216 kb)

Supplementary Figure 2

Quantitative analysis of protein dynamics of free CypA at 25°C. (PDF 197 kb)

Supplementary Figure 3

Pre-existing motions within the active site of CypA. (PDF 210 kb)

Supplementary Figure 4

Backbone amide chemical shift differences of CypA mutants. (PDF 258 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (DOC 30 kb)

Supplementary Tables

Supplementary Tables 1–4. (DOC 157 kb)

Supplementary Methods

Additional descriptions of methods used in this study. (DOC 29 kb)

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Eisenmesser, E., Millet, O., Labeikovsky, W. et al. Intrinsic dynamics of an enzyme underlies catalysis. Nature 438, 117–121 (2005). https://doi.org/10.1038/nature04105

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