Two-state allosteric behavior in a single-domain signaling protein.

Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.

[1]  A. Szabó,et al.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity , 1982 .

[2]  A. Palmer,et al.  Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. , 1995, Journal of molecular biology.

[3]  R. Nussinov,et al.  Folding and binding cascades: Dynamic landscapes and population shifts , 2008, Protein science : a publication of the Protein Society.

[4]  M. Akke,et al.  Structural dynamics in the C-terminal domain of calmodulin at low calcium levels. , 1999, Journal of molecular biology.

[5]  D. Koshland,et al.  Comparison of experimental binding data and theoretical models in proteins containing subunits. , 1966, Biochemistry.

[6]  F. Dahlquist,et al.  Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy. , 1994, Biochemistry.

[7]  Ann M Stock,et al.  Structural analysis of bacterial chemotaxis proteins: components of a dynamic signaling system. , 1998, Journal of structural biology.

[8]  J. Michael Schurr,et al.  A test of the model-free formulas. Effects of anisotropic rotational diffusion and dimerization. , 1994, Journal of magnetic resonance. Series B.

[9]  Victoria A. Feher,et al.  Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F , 1999, Nature.

[10]  M. Gerstein,et al.  The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework. , 2000, Nucleic acids research.

[11]  M. Eisenbach,et al.  Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor. , 1992, Biochemistry.

[12]  K. Dill.  Polymer principles and protein folding , 1999, Protein science : a publication of the Protein Society.

[13]  D. Torchia,et al.  Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution , 1999, Journal of Biomolecular NMR.

[14]  D. Kern,et al.  Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface. , 1997, Journal of molecular biology.

[15]  R. Bourret,et al.  Throwing the switch in bacterial chemotaxis. , 1999, Trends in microbiology.

[16]  S. Kustu,et al.  Unusual Oligomerization Required for Activity of NtrC, a Bacterial Enhancer-Binding Protein , 1997, Science.

[17]  J. Changeux,et al.  ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. , 1965, Journal of molecular biology.

[18]  A. Bax,et al.  Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements. , 1995, European journal of biochemistry.

[19]  S. Kustu,et al.  The bacterial enhancer-binding protein NtrC as a molecular machine. , 1998, Cold Spring Harbor symposia on quantitative biology.

[20]  G. Lipari.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules , 1982 .

[21]  M. Karplus,et al.  Protein Folding: A Perspective from Theory and Experiment. , 1998, Angewandte Chemie.

[22]  S. Kustu,et al.  Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution. , 1997, Journal of molecular biology.

[23]  A. Palmer,et al.  Probing molecular motion by NMR. , 1997, Current opinion in structural biology.

[24]  K. Volz,et al.  Structural conservation in the CheY superfamily. , 1993, Biochemistry.

[25]  Christopher D. Kroenke,et al.  The Static Magnetic Field Dependence of Chemical Exchange Linebroadening Defines the NMR Chemical Shift Time Scale , 2000 .

[26]  H. Frauenfelder,et al.  Conformational substates in proteins. , 1988, Annual review of biophysics and biophysical chemistry.

[27]  Brian F. Volkman,et al.  Structure of a transiently phosphorylated switch in bacterial signal transduction , 2000, Nature.

[28]  A. Palmer,et al.  Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13Cα nuclear spin relaxation , 1997, Journal of biomolecular NMR.

[29]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.