Abstract
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.
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References
Kuriyan, J. & Eisenberg, D. The origin of protein interactions and allostery in colocalization. Nature 450, 983–990 (2007)
Goodey, N. M. & Benkovic, S. J. Allosteric regulation and catalysis emerge via a common route. Nature Chem. Biol. 4, 474–482 (2008)
Smock, R. G. & Gierasch, L. M. Sending signals dynamically. Science 324, 198–203 (2009)
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)
Lee, J. et al. Surface sites for engineering allosteric control in proteins. Science 322, 438–442 (2008)
Changeux, J. P. & Edelstein, S. J. Allosteric mechanisms of signal transduction. Science 308, 1424–1428 (2005)
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)
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)
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)
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)
Berg, J. M., Tymoczko, J. L. & Stryer, L. Biochemistry 6th edn (Freeman, 2006)
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)
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)
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)
Mittermaier, A. & Kay, L. E. New tools provide new insights in NMR studies of protein dynamics. Science 312, 224–228 (2006)
Palmer, A. G. NMR characterization of the dynamics of biomacromolecules. Chem. Rev. 104, 3623–3640 (2004)
Kern, D. & Zuiderweg, E. R. The role of dynamics in allosteric regulation. Curr. Opin. Struct. Biol. 13, 748–757 (2003)
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)
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)
Cavanagh, J. & Akke, M. May the driving force be with you — whatever it is. Nature Struct. Biol. 7, 11–13 (2000)
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)
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)
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)
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)
Frederick, K. K., Marlow, M. S., Valentine, K. G. & Wand, A. J. Conformational entropy in molecular recognition by proteins. Nature 448, 325–329 (2007)
Popovych, N., Sun, S., Ebright, R. H. & Kalodimos, C. G. Dynamically driven protein allostery. Nature Struct. Mol. Biol. 13, 831–838 (2006)
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)
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)
Wand, A. J. Dynamic activation of protein function: a view emerging from NMR spectroscopy. Nature Struct. Biol. 8, 926–931 (2001)
Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007)
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)
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)
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)
Johnson, B. A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004)
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)
Palmer, A. G. III. ModelFree. 〈http://www.palmer.hs.columbia.edu/software/modelfree.html〉
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)
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)
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)
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)
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)
Palmer, A. G. III. quadric_diffusion. 〈http://www.palmer.hs.columbia.edu/software/quadric.html〉
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)
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)
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)
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)
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)
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)
Henzler-Wildman, K. A. et al. Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838–844 (2007)
Boehr, D. D., McElheny, D., Dyson, H. J. & Wright, P. E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313, 1638–1642 (2006)
Korzhnev, D. M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004)
Palmer, A. G. III. CPMGFit. 〈http://www.cumc.columbia.edu/dept/gsas/biochem/labs/palmer/software/cpmgfit.html〉
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)
Acknowledgements
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.
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Tzeng, SR., Kalodimos, C. Dynamic activation of an allosteric regulatory protein. Nature 462, 368–372 (2009). https://doi.org/10.1038/nature08560
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DOI: https://doi.org/10.1038/nature08560
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