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.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Walsh, D.A. & Van Patten, S.M. Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J. 8, 1227–1236 (1994).
Shabb, J.B. Physiological substrates of cAMP-dependent protein kinase. Chem. Rev. 101, 2381–2411 (2001).
Taylor, S.S. et al. PKA: A portrait of protein kinase dynamics. Biochim. Biophys. Acta 1697, 259–269 (2004).
Kornev, A.P. & Taylor, S.S. Defining the conserved internal architecture of a protein kinase. Biochim. Biophys. Acta 1804, 440–444 (2010).
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).
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).
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).
Popovych, N., Sun, S., Ebright, R.H. & Kalodimos, C.G. Dynamically driven protein allostery. Nat. Struct. Mol. Biol. 13, 831–838 (2006).
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).
Gsponer, J. et al. A coupled equilibrium shift mechanism in calmodulin-mediated signal transduction. Structure 16, 736–746 (2008).
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).
Mittag, T., Kay, L.E. & Forman-Kay, J.D. Protein dynamics and conformational disorder in molecular recognition. J. Mol. Recognit. 23, 105–116 (2009).
Tzeng, S.R. & Kalodimos, C.G. Dynamic activation of an allosteric regulatory protein. Nature 462, 368–372 (2009).
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).
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).
Henzler-Wildman, K.A. et al. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450, 913–916 (2007).
Traaseth, N.J. et al. Structural and dynamic basis of phospholamban and sarcolipin inhibition of ca(2+)-ATPase. Biochemistry 47, 3–13 (2008).
Adams, J.A. Kinetic and catalytic mechanisms of protein kinases. Chem. Rev. 101, 2271–2290 (2001).
Boehr, D.D., Nussinov, R. & Wright, P.E. The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789–796 (2009).
Mao, D.Y., Ceccarelli, D.F. & Sicheri, F. 'Unraveling the tail' of how SRPK1 phosphorylates ASF/SF2. Mol. Cell 29, 535–537 (2008).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Schwartz, S.D. & Schramm, V.L. Enzymatic transition states and dynamic motion in barrier crossing. Nat. Chem. Biol. 5, 551–558 (2009).
Smock, R.G. & Gierasch, L.M. Sending signals dynamically. Science 324, 198–203 (2009).
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).
Hammes, G.G. Multiple conformational changes in enzyme catalysis. Biochemistry 41, 8221–8228 (2002).
Hammes-Schiffer, S. & Benkovic, S.J. Relating protein motion to catalysis. Annu. Rev. Biochem. 75, 519–541 (2006).
Swain, J.F. & Gierasch, L.M. The changing landscape of protein allostery. Curr. Opin. Struct. Biol. 16, 102–108 (2006).
Das, R. et al. Dynamically driven ligand selectivity in cyclic nucleotide binding domains. J. Biol. Chem. 284, 23682–23696 (2009).
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).
Grünberg, R., Nilges, M. & Leckner, J. Flexibility and conformational entropy in protein-protein binding. Structure 14, 683–693 (2006).
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).
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).
Minor, W., Tomchick, D. & Otwinowski, Z. Strategies for macromolecular synchrotron crystallography. Structure 8, R105–R110 (2000).
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).
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).
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).
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).
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.
The authors declare no competing financial interests.
About this article
Cite this article
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
Everything you ever wanted to know about PKA regulation and its involvement in mammalian sperm capacitation
Molecular and Cellular Endocrinology (2020)
Biophysical Reviews (2020)
Proteins: Structure, Function, and Bioinformatics (2020)
Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A
Journal of Chemical Information and Modeling (2020)