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Visualization of transient encounter complexes in protein–protein association

Nature volume 444, pages 383–386 (2006)Cite this article

Abstract

Kinetic data on a number of protein–protein associations have provided evidence for the initial formation of a pre-equilibrium encounter complex that subsequently relaxes to the final stereospecific complex1. Site-directed mutagenesis2,3,4 and brownian dynamics simulations5,6,7 have suggested that the rate of association can be modulated by perturbations in charge distribution outside the direct interaction surfaces. Furthermore, rate enhancement through non-specific binding may occur by either a reduction in dimensionality8 or the presence of a short-range, non-specific attractive potential9. Here, using paramagnetic relaxation enhancement, we directly demonstrate the existence and visualize the distribution of an ensemble of transient, non-specific encounter complexes under equilibrium conditions for a relatively weak protein–protein complex between the amino-terminal domain of enzyme I and the phosphocarrier protein HPr. Neither the stereospecific complex10 alone nor any single alternative conformation can account fully for the intermolecular paramagnetic relaxation enhancement data. Restrained rigid-body simulated annealing refinement against the paramagnetic relaxation enhancement data enables us to obtain an atomic probability distribution map of the non-specific encounter complex ensemble that qualitatively correlates with the electrostatic surface potentials on the interacting proteins. Qualitatively similar results are presented for two other protein–protein complexes.

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Figure 1: Observed and calculated intermolecular PREs for the EIN–HPr complex.
Figure 2: Ensemble refinement and intermolecular PRE Q -factor.
Figure 3: Characterization of non-specific EIN–HPr encounter complexes.
Figure 4: Observed and calculated PRE 1HN-Γ2 values for the IIAMannitol–HPr complex.

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References

  1. Fersht, A. R. Structure and Mechanism in Protein Science: a Guide to Enzyme Catalysis and Protein Folding (Freeman & Co, New York, 1999)

  2. Schreiber, G. & Fersht, A. R. Rapid electrostatically assisted association of proteins. Nature Struct. Biol. 3, 427–431 (1996)

    Article  CAS  Google Scholar 

  3. Vijaykumar, M. et al. Electrostatic enhancement of diffusion-controlled protein–protein association: comparison of theory and experiment on barnase and barstar. J. Mol. Biol. 278, 1015–1024 (1998)

    Article  Google Scholar 

  4. Selzer, T., Albeck, S. & Schreiber, G. Rational design of faster associating and tighter binding protein complexes. Nature Struct. Biol. 7, 537–541 (2000)

    Article  CAS  PubMed  Google Scholar 

  5. Northrup, S. H., Boles, J. O. & Reynolds, J. C. L. Brownian dynamics of cytochrome c and cytochrome c peroxidase association. Science 241, 67–70 (1988)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Gabdoulline, R. R. & Wade, R. C. Biomolecular diffusional association. Curr. Opin. Struct. Biol. 12, 204–213 (2002)

    Article  CAS  Google Scholar 

  7. Spaar, A., Dammer, C., Gabdoulline, R. R., Wade, R. C. & Helms, V. Diffusional encounter of barnase and barstar. Biophys. J. 90, 1913–1924 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Adams, G. & Debruck, M. in Structural Chemistry and Molecular Biology (eds Rich, A. & Davidson, N.) 198–215 (Freeman & Co, San Francisco, CA, 1968)

  9. Zhou, H-X. & Szabo, A. Enhancement of association rates by nonspecific binding to DNA and cell membranes. Phys. Rev. Lett. 93, 178101 (2004)

    Article  ADS  Google Scholar 

  10. Garrett, D. S., Seok, Y-J., Peterkofsky, A., Gronenborn, A. M. & Clore, G. M. Solution structure of the 40,000 M r phosphoryl transfer complex between the N-terminal domain of enzyme I and HPr. Nature Struct. Biol. 6, 166–173 (1999)

    Article  CAS  PubMed  Google Scholar 

  11. Postma, P. W., Lengeler, J. W. & Jacobson, G. R. in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt, F. C.) 1149–1174 (ASM, Washington DC, 1996)

  12. Iwahara, J. & Clore, G. M. Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440, 1227–1230 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Iwahara, J., Schwieters, C. D. & Clore, G. M. Characterization of nonspecific protein–DNA interactions by 1H paramagnetic relaxation enhancement. J. Am. Chem. Soc. 126, 12800–12808 (2004)

    Article  CAS  Google Scholar 

  14. Ebright, Y. W., Chen, Y., Pendergrast, P. S. & Ebright, R. H. Incorporation of an EDTA–metal complex at a rationally selected site within a protein: application to EDTA–iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro. Biochemistry 31, 10664–10670 (1992)

    Article  CAS  PubMed  Google Scholar 

  15. Iwahara, J., Schwieters, C. D. & Clore, G. M. Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecules. J. Am. Chem. Soc. 126, 5879–5896 (2004)

    Article  CAS  Google Scholar 

  16. Donaldson, L. W. et al. Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy. J. Am. Chem. Soc. 123, 9843–9847 (2001)

    Article  CAS  Google Scholar 

  17. Pintacuda, G. & Otting, G. Identification of protein surfaces by NMR measurements with a paramagnetic Gd(iii) chelate. J. Am. Chem. Soc. 124, 372–373 (2002)

    Article  CAS  PubMed  Google Scholar 

  18. Schwieters, C. D., Kuszewski, J. J. & Clore, G. M. Using Xplor-NIH for NMR molecular structure determination. Prog. NMR Spectrosc. 48, 47–62 (2006)

    Article  CAS  Google Scholar 

  19. Brünger, A. T., Clore, G. M., Gronenborn, A. M. & Nilges, M. Assessing the quality of solution nuclear magnetic resonance structures by complete cross-validation. Science 261, 328–331 (1993)

    Article  ADS  PubMed  Google Scholar 

  20. Schwieters, C. D. & Clore, G. M. Reweighted atomic densities to represent ensembles of NMR structures. J. Biomol. NMR 23, 221–225 (2002)

    Article  CAS  PubMed  Google Scholar 

  21. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Jones, S. & Thornton, J. M. principles of protein–protein interactions. Proc. Natl Acad. Sci. USA 93, 13–20 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Cornilescu, G. et al. Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Eschrichia coli phosphotransferase system. J. Biol. Chem. 277, 42289–42298 (2002)

    Article  CAS  PubMed  Google Scholar 

  24. Williams, D. C., Cai, M., Suh, Y-J., Peterkofsky, A. & Clore, G. M. Solution NMR structure of the 48 kDa IIAMannose–HPr complex of the Escherichia coli mannose phosphotransferase system. J. Biol. Chem. 280, 20775–20784 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nilges, M., Gronenborn, A. M., Brünger, A. T. & Clore, G. M. Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints: application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2. Protein Eng. 2, 27–38 (1988)

    Article  CAS  PubMed  Google Scholar 

  26. Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improving the packing and accuracy of NMR structures with a pseudopotential for the radius of gyration. J. Am. Chem. Soc. 121, 2337–2338 (1999)

    Article  CAS  Google Scholar 

  27. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA, USA, 2002)

  28. Laskowski, R. A. SURFNET: a program for visualizing molecular surfaces, cavities and intermolecular interactions. J. Mol. Graph. 13, 323–330 (1995)

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank C. Schwieters, A. Szabo and C. Bewley for discussions; and C. Byeon for assistance with initial sample preparation. This work was supported by funds from the Intramural Program of the NIH, NIDDK and the AIDS Targeted Antiviral program of the Office of the Director of the NIH (to G.M.C.).

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Authors and Affiliations

  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Maryland, 20892-0520, Bethesda, USA

    Chun Tang, Junji Iwahara & G. Marius Clore

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  1. Chun Tang
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  2. Junji Iwahara
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Correspondence to G. Marius Clore.

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This file contains Supplementary Figures 1–6, Supplementary Methods, Supplementary Table and additional references. (PDF 453 kb)

Supplementary Movie 1

Movie representation of left-hand panels of Supplementary Figure 4a. (MOV 105070 kb)

Supplementary Movie 2

Movie representation of right-hand panels of Supplementary Figure 4a. (MOV 91291 kb)

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Tang, C., Iwahara, J. & Clore, G. Visualization of transient encounter complexes in protein–protein association. Nature 444, 383–386 (2006). https://doi.org/10.1038/nature05201

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