Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural landscape of isolated agonist-binding domains from single AMPA receptors

Nature Chemical Biology volume 7, pages 168–173 (2011)Cite this article

Subjects

Abstract

AMPA receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals, and thus it is important to understand the relationship between their chemical biology and their function. We used single-molecule fluorescence resonance energy transfer to examine the conformations explored by the agonist-binding domain of the AMPA receptor for wild-type and T686S mutant proteins. Each form of the agonist binding domain showed a dynamic, multistate sequential equilibrium, which could be identified only using wavelet shrinkage, a signal processing technique that removes experimental shot noise. These results illustrate that the extent of activation depends not on a rigid closed cleft but instead on the probability that a given subunit will occupy a closed-cleft conformation, which in turn is determined not only by the lowest energy state but also by the range of states that the protein explores.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal structure of GluA2-ABD.
Figure 2: Single-molecule smFRET before and after denoising.
Figure 3: Ensemble smFRET histograms.
Figure 4: Dwell-time histograms for all of the observed transitions between the adjacent four states identified from the glutamate-bound GluA2-ABD form.
Figure 5: Denoised ensemble smFRET histograms.
Figure 6: Average smFRET efficiency autocorrelation.

Similar content being viewed by others

References

  1. Keinänen, K. et al. A family of AMPA-selective glutamate receptors. Science 249, 556–560 (1990).

    Article  Google Scholar 

  2. Nakanishi, S. & Masu, M. Molecular diversity and functions of glutamate receptors. Annu. Rev. Biophys. Biomol. Struct. 23, 319–348 (1994).10.1146/annurev.bb.23.060194.001535

    Article  CAS  PubMed  Google Scholar 

  3. Dingledine, R., Borges, K., Bowie, D. & Traynelis, S.F. The glutamate receptor ion channels. Pharmacol. Rev. 51, 7–61 (1999).

    CAS  Google Scholar 

  4. Fleming, J.J. & England, P.M. AMPA receptors and synaptic plasticity: a chemist's perspective. Nat. Chem. Biol. 6, 89–97 (2010).

    Article  CAS  Google Scholar 

  5. Rosenmund, C., Stern-Bach, Y. & Stevens, C.F. The tetrameric structure of a glutamate receptor channel. Science 280, 1596–1599 (1998).

    Article  CAS  Google Scholar 

  6. Sobolevsky, A.I., Rosconi, M.P. & Gouaux, E. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462, 745–756 (2009).

    Article  CAS  Google Scholar 

  7. Armstrong, N. & Gouaux, E. Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluA2 agonist binding core. Neuron 28, 165–181 (2000).

    Article  CAS  Google Scholar 

  8. Armstrong, N., Mayer, M. & Gouaux, E. Tuning activation of the AMPA-sensitive GluA2 ion channel by genetic adjustment of agonist-induced conformational changes. Proc. Natl. Acad. Sci. USA 100, 5736–5741 (2003).

    Article  CAS  Google Scholar 

  9. Gouaux, E. Structure and function of AMPA receptors. J. Physiol. 554, 249–253 (2004).

    Article  CAS  Google Scholar 

  10. Armstrong, N., Sun, Y., Chen, G.-Q. & Gouaux, E. Structure of a glutamate-receptor agonist-binding core in complex with kainate. Nature 395, 913–917 (1998).

    Article  CAS  Google Scholar 

  11. Birdsey-Benson, A., Gill, A., Henderson, L.P. & Madden, D.R. Enhanced efficacy without further cleft closure: reevaluating twist as a source of agonist efficacy in AMPA receptors. J. Neurosci. 30, 1463–1470 (2010).

    Article  CAS  Google Scholar 

  12. Ahmed, A.H., Wang, Q., Sondermann, H. & Oswald, R.E. Structure of the S1S2 glutamate binding domain of GluR3. Proteins 75, 628–637 (2009).

    Article  CAS  Google Scholar 

  13. Maltsev, A.S., Ahmed, A.H., Fenwick, M.K., Jane, D.E. & Oswald, R.E. Mechanism of partial agonism at the GluA2 AMPA receptor: measurements of lobe orientation in solution. Biochemistry 47, 10600–10610 (2008).

    Article  CAS  Google Scholar 

  14. Ramanoudjame, G., Du, M., Mankiewicz, K.A. & Jayaraman, V. Allosteric mechanism in AMPA receptors: a FRET-based investigation of conformational changes. Proc. Natl. Acad. Sci. USA 103, 10473–10478 (2006).

    Article  CAS  Google Scholar 

  15. Robert, A., Armstrong, N., Gouaux, J.E. & Howe, J.R. AMPA receptor binding cleft mutations that alter affinity, efficacy, and recovery from desensitization. J. Neurosci. 25, 3752–3762 (2005).

    Article  CAS  Google Scholar 

  16. Lau, A.Y. & Roux, B. The free energy landscapes governing conformational changes in a glutamate receptor agonist-binding domain. Structure 15, 1203–1214 (2007).

    Article  CAS  Google Scholar 

  17. Li, C.-B., Yang, H. & Komatsuzaki, T. Multiscale complex network of protein conformational fluctuations in single-molecule time series. Proc. Natl. Acad. Sci. USA 105, 536–541 (2008).

    Article  CAS  Google Scholar 

  18. Schuler, B., Lipman, E.A. & Eaton, W.A. Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419, 743–747 (2002).

    Article  CAS  Google Scholar 

  19. Schuler, B. & Eaton, W.A. Protein folding studied by single-molecule FRET. Curr. Opin. Struct. Biol. 18, 16–26 (2008).

    Article  CAS  Google Scholar 

  20. Flynn, E.M., Hanson, J.A., Alber, T. & Yang, H. Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain. J. Am. Chem. Soc. 132, 4772–4780 (2010).

    Article  CAS  Google Scholar 

  21. Chung, H.S., Louis John, M. & Eaton William, A. Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments. Biophys. J. 98, 696–706 (2010).

    Article  CAS  Google Scholar 

  22. McKinney, S.A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).

    Article  CAS  Google Scholar 

  23. Talaga, D.S. Markov processes in single molecule fluorescence. Curr. Opin. Colloid Interface Sci. 12, 285–296 (2007).

    Article  CAS  Google Scholar 

  24. Li, C.-B., Yang, H. & Komatsuzaki, T. New quantification of local transition heterogeneity of multiscale complex networks constructed from single-molecule time series. J. Phys. Chem. B 113, 14732–14741 (2009).

    Article  CAS  Google Scholar 

  25. Taylor, J.N., Makarov, D.E. & Landes, C.F. Denoising single-molecule FRET trajectories with wavelets and Bayesian inference. Biophys. J. 98, 164–173 (2010).

    Article  CAS  Google Scholar 

  26. Taylor, J.N. & Landes, C.F. Improved resolution of complex single-molecule FRET systems via wavelet shrinkage. Journal of Physical Chemistry B, published online, doi:10.1021/jp1050707 (10 January 2011).

  27. Darugar, Q., Kim, H., Gorelick, R.J. & Landes, C.F. Human T-Cell lympotropic virus type 1 nucleocapsid protein-induced structural changes in transactivation response DNA measured by single molecule fluorescence resonance energy transfer. J. Virol. 82, 12164–12171 (2008).

    Article  CAS  Google Scholar 

  28. Taylor, J.N., Darugar, Q., Kourentzi, K., Willson, R.C. & Landes, C.F. Dynamics of an anti-VEGF aptamer: A single molecule study. Biochem. Biophys. Res. Commun. 373, 213–218 (2008).

    Article  Google Scholar 

  29. Mamonova, T., Yonkunas, M.J. & Kurnikova, M.G. Energetics of the cleft closing transition and the role of electrostatic interactions in conformational rearrangements of the glutamate receptor agonist binding domain. Biochemistry 47, 11077–11085 (2008).

    Article  CAS  Google Scholar 

  30. Benítez, J.J. et al. Probing transient copper chaperone-wilson disease protein interactions at the single-molecule level with nanovesicle trapping. J. Am. Chem. Soc. 130, 2446–2447 10.1021/ja7107867 (2008).

    Article  Google Scholar 

  31. Cheng, Q., Du, M., Ramanoudjame, G. & Jayaraman, V. Evolution of glutamate interactions during binding to a glutamate receptor. Nat. Chem. Biol. 1, 329–332 (2005).

    Article  CAS  Google Scholar 

  32. Ahmed, A.H., Loh, A.P., Jane, D.E. & Oswald, R.E. Dynamics of the S1S2 glutamate binding domain of GluA2 measured using 19F NMR spectroscopy. J. Biol. Chem. 282, 12773–12784 (2007).

    Article  CAS  Google Scholar 

  33. Fenwick, M.K. & Oswald, R.E. On the mechanisms of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor binding to glutamate and kainate. J. Biol. Chem. 285, 12334–12343 (2010).

    Article  CAS  Google Scholar 

  34. McFeeters, R.L. & Oswald, R.E. Structural mobility of the extracellular agonist-binding core of an ionotropic glutamate receptor. Analysis of NMR relaxation dynamics. Biochemistry 41, 10472–10481 (2002).

    Article  CAS  Google Scholar 

  35. Robert, A. & Howe, J.R. How AMPA receptor desensitization depends on receptor occupancy. J. Neurosci. 23, 847–858 (2003).

    Article  CAS  Google Scholar 

  36. Zhang, W., Cho, Y., Lolis, E. & Howe, J.R. Structural and single-channel results indicate that the rates of agonist binding domain closing and opening directly impact AMPA receptor gating. J. Neurosci. 28, 932–943 (2008).

    Article  Google Scholar 

  37. Makarov, D.E. & Metiu, H. A model for the kinetics of protein folding: kinetic Monte Carlo simulations and analytical results. J. Chem. Phys. 116, 5205–5216 (2002).

    Article  CAS  Google Scholar 

  38. Fichthorn, K.A. & Weinberg, W.H. Theoretical foundations of dynamical Monte Carlo simulations. J. Chem. Phys. 95, 1090–1096 (1991).

    Article  CAS  Google Scholar 

  39. Metiu, H., Lu, Y.-T. & Zhang, Z. Epitaxial growth and the art of computer simulations. Science 255, 1088–1092 (1992).

    Article  CAS  Google Scholar 

  40. Voter, A.F. Classically exact overlayer dynamics: diffusion of rhodium clusters on Rh(100). Phys. Rev. B Condens. Matter 34, 6819–6929 (1986).

    Article  CAS  Google Scholar 

  41. Madden, D.R., Armstrong, N., Svergun, D., Perez, J. & Vachette, P. Solution X-ray scattering evidence for agonist- and antagonist-induced modulation of cleft closure in a glutamate receptor agonist-binding domain. J. Biol. Chem. 280, 23637–23642 (2005).

    Article  CAS  Google Scholar 

  42. Ha, T. et al. Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl. Acad. Sci. USA 96, 893–898 (1999).

    Article  CAS  Google Scholar 

  43. Landes, C.F., Zeng, Y., Liu, H.W., Musier-Forsyth, K. & Barbara, P.F. Single-molecule study of the inhibition of HIV-1 transactivation response region DNA/DNA annealing by argininamide. J. Am. Chem. Soc. 129, 10181–10188 (2007).

    Article  CAS  Google Scholar 

  44. Cosa, G. et al. Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein. Biophys. J. 87, 2759–2767 (2004).

    Article  CAS  Google Scholar 

  45. Hanson, J.A. et al. Illuminating the mechanistic roles of enzyme conformational dynamics. Proc. Natl. Acad. Sci. USA 104, 18055–18060 (2007).

    Article  CAS  Google Scholar 

  46. Zeng, Y. et al. Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer. Proc. Natl. Acad. Sci. USA 104, 12651–12656 10.1073/pnas.0700350104 (2007).

    Article  CAS  Google Scholar 

  47. Cordes, T., Vogelsang, J. & Tinnefeld, P. On the mechanism of Trolox as antiblinking and antibleaching reagent. J. Am. Chem. Soc. 131, 5018–5019 (2009).

    Article  CAS  Google Scholar 

  48. Rasnik, I., McKinney, S.A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

C.F.L. thanks the Norman Hackerman Welch Young Investigator Program at Rice University. We acknowledge the Donors of the American Chemical Society Petroleum Research Fund for partial support of this research (to C.F.L.). This work was supported by US National Institutes of Health Grant R01GM073102 (to V.J.) and American Heart Association Grant 0855081F (V.J.).We also thank H. Yang and R. Goldsmith for suggestions.

Author information

Authors and Affiliations

  1. Department of Chemistry, Rice University, Houston, Texas, USA

    Christy F Landes, J Nick Taylor & Ferandre Salatan

  2. Department of Biochemistry and Molecular Biology, Center for Membrane Biology, Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, Texas, USA

    Anu Rambhadran & Vasanthi Jayaraman

Authors
  1. Christy F Landes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anu Rambhadran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Nick Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ferandre Salatan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vasanthi Jayaraman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.F.L. and V.J. directed the research and wrote the manuscript. A.R. prepared and purified the protein samples and performed single-molecule experiments. J.N.T. analyzed the single-molecule data and prepared figures. F.S. performed single-molecule experiments and analyzed data.

Corresponding authors

Correspondence to Christy F Landes or Vasanthi Jayaraman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–13 and Supplementary Tables 1–4 (PDF 1307 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Landes, C., Rambhadran, A., Taylor, J. et al. Structural landscape of isolated agonist-binding domains from single AMPA receptors. Nat Chem Biol 7, 168–173 (2011). https://doi.org/10.1038/nchembio.523

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.523

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing