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Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Nature volume 440, pages 1227–1230 (2006)Cite this article

Abstract

Macromolecular complex formation is governed by two opposing constraints of specificity and speed1,2. Kinetic3,4,5,6 and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process1,7 or by the creation of a short-range attractive potential around the target site2. This implies the existence of transient intermediates involving non-specific binding modes. Here we show that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence of, and investigating the nature of, low population transient intermediates under equilibrium conditions. Applying this approach, we characterize the search process whereby a sequence-specific transcription factor (the homeodomain of HOXD9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. The intermolecular PRE method is general and can be readily applied to investigations of transient intermediates in many other macromolecular binding processes.

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Figure 1: Intermolecular PRE in an exchanging system.
Figure 2: Intermolecular PRE for the HOXD9 homeodomain/DNA complex in slow and fast exchange.
Figure 3: Summary of the intermolecular PRE profiles arising from dT-EDTA-Mn 2+ at sites 1 to 4 for the HOXD9 homeodomain/DNA complex in the slow and fast exchange regimes.
Figure 4: Intermolecular PRE arises from inter- and intramolecular translocation processes.

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References

  1. Berg, O. G. & von Hippel, P. H. Diffusion-controlled macromolecular interactions. Annu. Rev. Biophys. Biophys. Chem. 14, 131–160 (1985)

    Article  CAS  PubMed  Google Scholar 

  2. 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  PubMed  Google Scholar 

  3. von Hippel, P. H. & Berg, O. G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989)

    CAS  PubMed  Google Scholar 

  4. Halford, S. E. & Marko, J. F. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res. 32, 3040–3052 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Vijayakumar, 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  CAS  PubMed  Google Scholar 

  7. Adam, G. & Delbruck, M. in Structural Chemistry and Molecular Biology (eds Rich, A. & Davidson, N.) 198–215 (Freeman & Co., San Francisco, 1968)

    Google Scholar 

  8. Solomon, I. Relaxation processes in a system of two spins. Phys. Rev. 99, 559–565 (1955)

    Article  ADS  CAS  Google Scholar 

  9. Bloembergen, N. & Morgan, L. O. Proton relaxation times in paramagnetic solutions. Effects of election spin relaxation. J. Chem. Phys. 34, 842–850 (1961)

    Article  ADS  CAS  Google Scholar 

  10. 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 macromolecule. J. Am. Chem. Soc. 126, 5879–5896 (2004)

    Article  CAS  PubMed  Google Scholar 

  11. 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  PubMed  Google Scholar 

  12. Iwahara, J., Anderson, D. E., Murphy, E. C. & Clore, G. M. EDTA-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation enhancement. J. Am. Chem. Soc. 125, 6634–6635 (2003)

    Article  CAS  PubMed  Google Scholar 

  13. Gehring, W. J. et al. Homeodomain-DNA recognition. Cell 78, 211–223 (1994)

    Article  CAS  PubMed  Google Scholar 

  14. Billeter, M. et al. Determination of the nuclear magnetic resonance solution structure of an Antennapedia homeodomain-DNA complex. J. Mol. Biol. 234, 1084–1093 (1993)

    Article  CAS  PubMed  Google Scholar 

  15. Fraenkel, E. & Pabo, C. O. Comparison of X-ray and NMR structures for the Antennapedia homeodomain-DNA complex. Nature Struct. Biol. 5, 692–697 (1998)

    Article  CAS  PubMed  Google Scholar 

  16. Clore, G. M. & Garrett, D. S. R-factor, free R and complete cross-validation for dipolar coupling refinement of NMR structures. J. Am. Chem. Soc. 121, 9008–9012 (1999)

    Article  CAS  Google Scholar 

  17. Iwahara, J. & Clore, G. M. Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy. J. Am. Chem. Soc. 128, 404–405 (2006)

    Article  CAS  PubMed  Google Scholar 

  18. Affolter, M., Percival-Smith, A., Müller, M., Leupin, W. & Gehring, W. J. DNA binding properties of the purified Antennapedia homeodomain. Proc. Natl Acad. Sci. USA 87, 4093–4097 (1990)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Catron, K. M., Iler, N. & Abate, C. Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins. Mol. Cell. Biol. 13, 2354–2365 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Misteli, T. Protein dynamics: implications for nuclear architecture and gene expression. Science 291, 843–847 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Lewin, B. Genes VII (Oxford Univ. Press, Oxford, 2000)

    Google Scholar 

  22. Gillespie, J. R. & Shortle, D. Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. Paramagnetic relaxation enhancement by nitroxide spin labels. J. Mol. Biol. 268, 158–169 (1997)

    Article  CAS  PubMed  Google Scholar 

  23. Dvoretsky, A., Gaponenko, V. & Rosevear, P. R. Derivation of structural restraints using a thiol-reactive chelator. FEBS Lett. 528, 189–192 (2002)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. 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  PubMed  Google Scholar 

  26. Schwieters, C. D., Kuszewski, J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  27. McConnell, H. M. Reaction rates by nuclear magnetic resonance. J. Chem. Phys. 28, 430–431 (1958)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Intramural Program of the NIH, the NIDDK, and in part by the AIDS Targeted Antiviral Program of the Office of the Director of the NIH (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, Bethesda, Maryland, 20892-0520, USA

    Junji Iwahara & G. Marius Clore

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  1. Junji Iwahara
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Correspondence to G. Marius Clore.

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Supplementary Notes

This file contains details of lineshape simulations, residual dipolar coupling data, NMR exchange experiments and PRE profiles to be linked to the online version of the paper. (PDF 1671 kb)

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Iwahara, J., Clore, G. Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440, 1227–1230 (2006). https://doi.org/10.1038/nature04673

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