Dynamics connect substrate recognition to catalysis in protein kinase A

A Corrigendum to this article was published on 18 April 2011

This article has been updated


Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, X-ray crystallography and thermodynamic measurements, we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously and priming it for catalysis. The formation of the ternary complex is entropically driven, and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicates that a conformational selection rather than an induced-fit mechanism governs substrate recognition.

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Figure 1: X-ray crystal structure of the PKA-C ternary complex containing AMP-PNP and PLN1–19.
Figure 2: Details of the interaction of isotopically labeled PLN1–20 with PKA-C.
Figure 3: Mapping of the backbone amide dynamics of PKA-C from the apo to ternary complex.
Figure 4: Opening and closing of the enzyme active site cleft.
Figure 5: Model for the mechanism of the formation of a catalytically competent ternary complex.

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Change history

  • 18 April 2011

    In the version of this article initially published, the authors incorrectly acknowledged funding from the US National Institutes of Health, GM64742. The correct grant number should be GM072701. The error has been corrected in the HTML and PDF versions of the article.


  1. 1

    Walsh, D.A. & Van Patten, S.M. Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J. 8, 1227–1236 (1994).

    CAS  Article  Google Scholar 

  2. 2

    Shabb, J.B. Physiological substrates of cAMP-dependent protein kinase. Chem. Rev. 101, 2381–2411 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Taylor, S.S. et al. PKA: A portrait of protein kinase dynamics. Biochim. Biophys. Acta 1697, 259–269 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Kornev, A.P. & Taylor, S.S. Defining the conserved internal architecture of a protein kinase. Biochim. Biophys. Acta 1804, 440–444 (2010).

    CAS  Article  Google Scholar 

  5. 5

    Johnson, D.A., Akamine, P., Radzio-Andzelm, E., Madhusudan, M. & Taylor, S.S. Dynamics of cAMP-dependent protein kinase. Chem. Rev. 101, 2243–2270 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Vajpai, N. et al. Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of imatinib/nilotinib and dasatinib. J. Biol. Chem. 283, 18292–18302 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Jarymowycz, V.A. & Stone, M.J. Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences. Chem. Rev. 106, 1624–1671 (2006).

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

    Marlow, M.S., Dogan, J., Frederick, K.K., Valentine, K.G. & Wand, A.J. The role of conformational entropy in molecular recognition by calmodulin. Nat. Chem. Biol. 6, 352–358 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Gsponer, J. et al. A coupled equilibrium shift mechanism in calmodulin-mediated signal transduction. Structure 16, 736–746 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Yao, X., Rosen, M.K. & Gardner, K.H. Estimation of the available free energy in a LOV2-J alpha photoswitch. Nat. Chem. Biol. 4, 491–497 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Mittag, T., Kay, L.E. & Forman-Kay, J.D. Protein dynamics and conformational disorder in molecular recognition. J. Mol. Recognit. 23, 105–116 (2009).

    Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

    Henzler-Wildman, K.A. et al. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450, 913–916 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Traaseth, N.J. et al. Structural and dynamic basis of phospholamban and sarcolipin inhibition of ca(2+)-ATPase. Biochemistry 47, 3–13 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Adams, J.A. Kinetic and catalytic mechanisms of protein kinases. Chem. Rev. 101, 2271–2290 (2001).

    CAS  Article  Google Scholar 

  19. 19

    Boehr, D.D., Nussinov, R. & Wright, P.E. The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789–796 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Mao, D.Y., Ceccarelli, D.F. & Sicheri, F. 'Unraveling the tail' of how SRPK1 phosphorylates ASF/SF2. Mol. Cell 29, 535–537 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Masterson, L.R. et al. Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis. Protein Expr. Purif. 64, 231–236 (2009).

    CAS  Article  Google Scholar 

  22. 22

    Masterson, L.R., Mascioni, A., Traaseth, N.J., Taylor, S.S. & Veglia, G. Allosteric cooperativity in protein kinase A. Proc. Natl. Acad. Sci. USA 105, 506–511 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Kay, L.E., Torchia, D.A. & Bax, A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. Biochemistry 28, 8972–8979 (1989).

    CAS  Article  Google Scholar 

  24. 24

    Fenwick, M.K. & Oswald, R.E. NMR spectroscopy of the ligand-binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists. J. Mol. Biol. 378, 673–685 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Wang, C., Rance, M. & Palmer, A.G. 3rd Mapping chemical exchange in proteins with MW > 50 kD. J. Am. Chem. Soc. 125, 8968–8969 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Shan, Y. et al. A conserved protonation-dependent switch controls drug binding in the abl kinase. Proc. Natl. Acad. Sci. USA 106, 139–144 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Lew, J., Taylor, S.S. & Adams, J.A. Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: A transient kinetic study using stopped-flow fluorescence spectroscopy. Biochemistry 36, 6717–6724 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Massi, F., Wang, C. & Palmer, A.G. 3rd Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase. Biochemistry 45, 10787–10794 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Li, F., Juliano, C., Gorfain, E., Taylor, S.S. & Johnson, D.A. Evidence for an internal entropy contributin to phosphoryl transfer: A study of domain clossure, backbone flexibility, and the catalytic cycle of cAMP-dependent protein kinase. J. Mol. Biol. 315, 459–469 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Kim, C., Cheng, C.Y., Saldanha, S.A. & Taylor, S.S. PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation. Cell 130, 1032–1043 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Yang, J. et al. Allosteric network of cAMP-dependent protein kinase revealed by mutation of Tyr204 in the P+1 loop. J. Mol. Biol. 346, 191–201 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Hyeon, C., Jennings, P.A., Adams, J.A. & Onuchic, J.N. Ligand-induced global transitions in the catalytic domain of protein kinase A. Proc. Natl. Acad. Sci. USA 106, 3023–3028 (2009).

    CAS  Article  Google Scholar 

  33. 33

    Wu, J. et al. Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix. Protein Sci. 14, 2871–2879 (2005).

    CAS  Article  Google Scholar 

  34. 34

    Kamerlin, S.C. & Warshel, A. At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis? Proteins 78, 1339–1375 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Schwartz, S.D. & Schramm, V.L. Enzymatic transition states and dynamic motion in barrier crossing. Nat. Chem. Biol. 5, 551–558 (2009).

    CAS  Article  Google Scholar 

  36. 36

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

    CAS  Article  Google Scholar 

  37. 37

    Kumar, S., Ma, B., Tsai, C.J., Sinha, N. & Nussinov, R. Folding and binding cascades: Dynamic landscapes and population shifts. Protein Sci. 9, 10–19 (2000).

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

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

    CAS  Article  Google Scholar 

  40. 40

    Swain, J.F. & Gierasch, L.M. The changing landscape of protein allostery. Curr. Opin. Struct. Biol. 16, 102–108 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Das, R. et al. Dynamically driven ligand selectivity in cyclic nucleotide binding domains. J. Biol. Chem. 284, 23682–23696 (2009).

    CAS  Article  Google Scholar 

  42. 42

    Müller, C.W., Schlauderer, G.J., Reinstein, J. & Schulz, G.E. Adenylate kinase motions during catalysis: An energetic counterweight balancing substrate binding. Structure 4, 147–156 (1996).

    Article  Google Scholar 

  43. 43

    Grünberg, R., Nilges, M. & Leckner, J. Flexibility and conformational entropy in protein-protein binding. Structure 14, 683–693 (2006).

    Article  Google Scholar 

  44. 44

    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  Article  Google Scholar 

  45. 45

    Masterson, L.R. et al. Backbone NMR resonance assignment of the catalytic subunit of cAMP-dependent protein kinase A in complex with AMP-PNP. Biomol. NMR Assign. 3, 115–117 (2009).

    CAS  Article  Google Scholar 

  46. 46

    Minor, W., Tomchick, D. & Otwinowski, Z. Strategies for macromolecular synchrotron crystallography. Structure 8, R105–R110 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. & Bax, A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Article  Google Scholar 

  48. 48

    Farrow, N.A. et al. Backbone dynamics of a free and phosphopeptide-complexed src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994).

    CAS  Article  Google Scholar 

  49. 49

    Pervushin, K., Riek, R., Wider, G. & Wuthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl. Acad. Sci. USA 94, 12366–12371 (1997).

    CAS  Article  Google Scholar 

  50. 50

    Tjandra, N., Wingfield, P., Stahl, S. & Bax, A. Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields. J. Biomol. NMR 8, 273–284 (1996).

    CAS  Article  Google Scholar 

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This work was supported by the US National Institutes of Health (GM072701 and HL080081 to G.V. and GM19301 to S.S.T.). NMR data were collected at the National Magnetic Resonance Facility at Madison (NMRFAM) (US National Institutes of Health: P41RR02301, P41GM66326, RR02781 and RR08438; US National Science Foundation: DMB-8415048, OIA-9977486 and BIR-9214394) and the University of Minnesota NMR Facility (US National Science Foundation BIR-961477). We thank J.P. Loria (Yale University) for providing the TROSY Hahn echo pulse sequence, and we would also like to thank J.P. Loria, E.E. Metcalfe and G. Melacini for critical analysis of the paper.

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L.R.M., C.C., T.Y., M.T., A.K., S.S.T. and G.V. designed experiments, analyzed data and wrote the paper, and L.R.M., C.C., T.Y. and M.T. performed experiments.

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Correspondence to Susan S Taylor or Gianluigi Veglia.

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Supplementary Methods, Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1 & 2 (PDF 5952 kb)

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Masterson, L., Cheng, C., Yu, T. et al. Dynamics connect substrate recognition to catalysis in protein kinase A. Nat Chem Biol 6, 821–828 (2010). https://doi.org/10.1038/nchembio.452

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