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.

Dynamic activation of an allosteric regulatory protein


Allosteric regulation is used as a very efficient mechanism to control protein activity in most biological processes, including signal transduction, metabolism, catalysis and gene regulation1,2,3,4,5,6. Allosteric proteins can exist in several conformational states with distinct binding or enzymatic activity. Effectors are considered to function in a purely structural manner by selectively stabilizing a specific conformational state, thereby regulating protein activity. Here we show that allosteric proteins can be regulated predominantly by changes in their structural dynamics. We have used NMR spectroscopy and isothermal titration calorimetry to characterize cyclic AMP (cAMP) binding to the catabolite activator protein (CAP), a transcriptional activator that has been a prototype for understanding effector-mediated allosteric control of protein activity7. cAMP switches CAP from the ‘off’ state (inactive), which binds DNA weakly and non-specifically, to the ‘on’ state (active), which binds DNA strongly and specifically. In contrast, cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation; yet, cAMP binding to CAP-S62F strongly activates the protein for DNA binding. NMR and thermodynamic analyses show that despite the fact that CAP-S62F-cAMP2 adopts the inactive conformation, its strong binding to DNA is driven by a large conformational entropy originating in enhanced protein motions induced by DNA binding. The results provide strong evidence that changes in protein motions may activate allosteric proteins that are otherwise structurally inactive.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Conformational states of CAP and effect of cAMP binding assessed by NMR.
Figure 2: Energetics of CAP interaction with DNA.
Figure 3: CAP-S62F-cAMP 2 visits an active, low-populated conformational state.
Figure 4: Reaction pathways and energetics for cAMP-mediated CAP activation and DNA binding.


  1. 1

    Kuriyan, J. & Eisenberg, D. The origin of protein interactions and allostery in colocalization. Nature 450, 983–990 (2007)

    CAS  Google Scholar 

  2. 2

    Goodey, N. M. & Benkovic, S. J. Allosteric regulation and catalysis emerge via a common route. Nature Chem. Biol. 4, 474–482 (2008)

    CAS  Google Scholar 

  3. 3

    Smock, R. G. & Gierasch, L. M. Sending signals dynamically. Science 324, 198–203 (2009)

    CAS  Google Scholar 

  4. 4

    del Sol, A., Tsai, C. J., Ma, B. & Nussinov, R. The origin of allosteric functional modulation: multiple pre-existing pathways. Structure 17, 1042–1050 (2009)

    CAS  Google Scholar 

  5. 5

    Lee, J. et al. Surface sites for engineering allosteric control in proteins. Science 322, 438–442 (2008)

    CAS  Google Scholar 

  6. 6

    Changeux, J. P. & Edelstein, S. J. Allosteric mechanisms of signal transduction. Science 308, 1424–1428 (2005)

    CAS  Google Scholar 

  7. 7

    Won, H. S., Lee, Y. S., Lee, S. H. & Lee, B. J. Structural overview on the allosteric activation of cyclic AMP receptor protein. Biochim. Biophys. Acta 1794, 1299–1308 (2009)

    CAS  Google Scholar 

  8. 8

    Schultz, S. C., Shields, G. C. & Steitz, T. A. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science 253, 1001–1007 (1991)

    CAS  Google Scholar 

  9. 9

    Popovych, N., Tzeng, S. R., Tonelli, M., Ebright, R. H. & Kalodimos, C. G. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein. Proc. Natl Acad. Sci. USA 106, 6927–6932 (2009)

    CAS  Google Scholar 

  10. 10

    Passner, J. M., Schultz, S. C. & Steitz, T. A. Modeling the cAMP-induced allosteric transition using the crystal structure of CAP-cAMP at 2.1 Å resolution. J. Mol. Biol. 304, 847–859 (2000)

    CAS  Google Scholar 

  11. 11

    Berg, J. M., Tymoczko, J. L. & Stryer, L. Biochemistry 6th edn (Freeman, 2006)

    Google Scholar 

  12. 12

    Dai, J., Lin, S. H., Kemmis, C., Chin, A. J. & Lee, J. C. Interplay between site-specific mutations and cyclic nucleotides in modulating DNA recognition by Escherichia coli cyclic AMP receptor protein. Biochemistry 43, 8901–8910 (2004)

    CAS  Google Scholar 

  13. 13

    Baichoo, N. & Heyduk, T. Mapping conformational changes in a protein: application of a protein footprinting technique to cAMP-induced conformational changes in cAMP receptor protein. Biochemistry 36, 10830–10836 (1997)

    CAS  Google Scholar 

  14. 14

    Aiba, H., Nakamura, T., Mitani, H. & Mori, H. Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli . EMBO J. 4, 3329–3332 (1985)

    CAS  Google Scholar 

  15. 15

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

    CAS  Google Scholar 

  16. 16

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

    CAS  Google Scholar 

  17. 17

    Kern, D. & Zuiderweg, E. R. The role of dynamics in allosteric regulation. Curr. Opin. Struct. Biol. 13, 748–757 (2003)

    CAS  Google Scholar 

  18. 18

    Akke, M., Bruschweiler, R. & Palmer, A. G. NMR order parameters and free energy: an analytical approach and its application to cooperative Ca2+ binding by calbindin D9k . J. Am. Chem. Soc. 115, 9832–9833 (1993)

    CAS  Google Scholar 

  19. 19

    Yang, D. & Kay, L. E. Contributions to conformational entropy arising from bond vector fluctuations measured from NMR-derived order parameters: application to protein folding. J. Mol. Biol. 263, 369–382 (1996)

    CAS  Google Scholar 

  20. 20

    Cavanagh, J. & Akke, M. May the driving force be with you — whatever it is. Nature Struct. Biol. 7, 11–13 (2000)

    CAS  Google Scholar 

  21. 21

    Zhang, F. & Bruschweiler, R. Contact model for the prediction of NMR N-H order parameters in globular proteins. J. Am. Chem. Soc. 124, 12654–12655 (2002)

    CAS  Google Scholar 

  22. 22

    Kay, L. E., Muhandiram, D. R., Wolf, G., Shoelson, S. E. & Forman-Kay, J. D. Correlation between binding and dynamics at SH2 domain interfaces. Nature Struct. Biol. 5, 156–163 (1998)

    CAS  Google Scholar 

  23. 23

    Bracken, C., Carr, P. A., Cavanagh, J. & Palmer, A. G. Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA. J. Mol. Biol. 285, 2133–2146 (1999)

    CAS  Google Scholar 

  24. 24

    Mauldin, R. V., Carroll, M. J. & Lee, A. L. Dynamic dysfunction in dihydrofolate reductase results from antifolate drug binding: modulation of dynamics within a structural state. Structure 17, 386–394 (2009)

    CAS  Google Scholar 

  25. 25

    Frederick, K. K., Marlow, M. S., Valentine, K. G. & Wand, A. J. Conformational entropy in molecular recognition by proteins. Nature 448, 325–329 (2007)

    CAS  Google Scholar 

  26. 26

    Popovych, N., Sun, S., Ebright, R. H. & Kalodimos, C. G. Dynamically driven protein allostery. Nature Struct. Mol. Biol. 13, 831–838 (2006)

    CAS  Google Scholar 

  27. 27

    MacRaild, C. A., Daranas, A. H., Bronowska, A. & Homans, S. W. Global changes in local protein dynamics reduce the entropic cost of carbohydrate binding in the arabinose-binding protein. J. Mol. Biol. 368, 822–832 (2007)

    CAS  Google Scholar 

  28. 28

    Kim, J., Adhya, S. & Garges, S. Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation. Proc. Natl Acad. Sci. USA 89, 9700–9704 (1992)

    CAS  Google Scholar 

  29. 29

    Wand, A. J. Dynamic activation of protein function: a view emerging from NMR spectroscopy. Nature Struct. Biol. 8, 926–931 (2001)

    CAS  Google Scholar 

  30. 30

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

    CAS  Google Scholar 

  31. 31

    Parkinson, G. et al. Structure of the CAP-DNA complex at 2.5 Å resolution: a complete picture of the protein-DNA interface. J. Mol. Biol. 260, 395–408 (1996)

    CAS  Google Scholar 

  32. 32

    Takeuchi, K., Ng, E., Malia, T. J. & Wagner, G. 1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins. J. Biomol. NMR 38, 89–98 (2007)

    CAS  Google Scholar 

  33. 33

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

    CAS  Google Scholar 

  34. 34

    Johnson, B. A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004)

    CAS  Google Scholar 

  35. 35

    Evenäs, J. et al. Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy. J. Mol. Biol. 309, 961–974 (2001)

    Google Scholar 

  36. 36

    Palmer, A. G. III. ModelFree. 〈

  37. 37

    Cole, R. & Loria, J. P. FAST-Modelfree: a program for rapid automated analysis of solution NMR spin-relaxation data. J. Biomol. NMR 26, 203–213 (2003)

    CAS  Google Scholar 

  38. 38

    d'Auvergne, E. J. & Gooley, P. R. Optimisation of NMR dynamic models I. Minimisation algorithms and their performance within the model-free and Brownian rotational diffusion spaces. J. Biomol. NMR 40, 107–119 (2008)

    CAS  Google Scholar 

  39. 39

    Lipari, G. & Szabo, A. Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559 (1982)

    CAS  Google Scholar 

  40. 40

    Tjandra, N., Feller, S. E., Pastor, R. W. & Bax, A. Rotational diffusion anisotropy of human ubiquitin from 15N NMR relaxation. J. Am. Chem. Soc. 117, 12562–12566 (1995)

    CAS  Google Scholar 

  41. 41

    Hwang, P. M., Skrynnikov, N. R. & Kay, L. E. Domain orientation in beta-cyclodextrin-loaded maltose binding protein: diffusion anisotropy measurements confirm the results of a dipolar coupling study. J. Biomol. NMR 20, 83–88 (2001)

    CAS  Google Scholar 

  42. 42

    Palmer, A. G. III. quadric_diffusion. 〈

  43. 43

    Dosset, P., Hus, J. C., Blackledge, M. & Marion, D. Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data. J. Biomol. NMR 16, 23–28 (2000)

    CAS  Google Scholar 

  44. 44

    Mandel, A. M., Akke, M. & Palmer, A. G. Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J. Mol. Biol. 246, 144–163 (1995)

    CAS  Google Scholar 

  45. 45

    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  Google Scholar 

  46. 46

    Mulder, F. 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  Google Scholar 

  47. 47

    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)

    CAS  Google Scholar 

  48. 48

    Watt, E. D., Shimada, H., Kovrigin, E. L. & Loria, J. P. The mechanism of rate-limiting motions in enzyme function. Proc. Natl Acad. Sci. USA 104, 11981–11986 (2007)

    CAS  Google Scholar 

  49. 49

    Henzler-Wildman, K. A. et al. Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838–844 (2007)

    CAS  Google Scholar 

  50. 50

    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  Google Scholar 

  51. 51

    Korzhnev, D. M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004)

    CAS  Google Scholar 

  52. 52

    Palmer, A. G. III. CPMGFit. 〈

  53. 53

    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  Google Scholar 

Download references


We are grateful to L. Kay for critical reading of the manuscript and valuable suggestions. We thank R. H. Ebright and Y. Ebright for providing the DNA fragment and N. Popovych for her help with the preparation of some CAP mutants. This work was supported by National Science Foundation (NSF) grant MCB618259 to C.G.K.

Author Contributions C.G.K. conceived the project. S.-R.T. and C.G.K. designed the experiments. S.-R.T. performed all experiments. S.-R.T. and C.G.K. analysed and interpreted data and wrote the manuscript.

Author information



Corresponding author

Correspondence to Charalampos G. Kalodimos.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S16 with Legends and Supplementary References. (PDF 3642 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tzeng, SR., Kalodimos, C. Dynamic activation of an allosteric regulatory protein. Nature 462, 368–372 (2009).

Download citation

Further reading


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