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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

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

  2. 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

  3. 3

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

  4. 4

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

  5. 5

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

  6. 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).

  7. 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).

  8. 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).

  9. 9

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

  10. 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).

  11. 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).

  12. 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).

  13. 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).

  14. 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).

  15. 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).

  16. 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).

  17. 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).

  18. 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).

  19. 19

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

  20. 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).

  21. 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).

  22. 22

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

  23. 23

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

  24. 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).

  25. 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).

  26. 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. 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).

  28. 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).

  29. 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).

  30. 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).

  31. 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).

  32. 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).

  33. 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).

  34. 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).

  35. 35

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

  36. 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).

  37. 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).

  38. 38

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

  39. 39

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

  40. 40

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

  41. 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).

  42. 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).

  43. 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).

  44. 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).

  45. 45

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

  46. 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).

  47. 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).

  48. 48

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

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

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.

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

Further reading