Luminescence Resonance Energy Transfer Investigation of ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 40, pp. 27074 –27078, October 3, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a Kainate Receptor*□ S
Received for publication, July 2, 2008, and in revised form, July 23, 2008 Published, JBC Papers in Press, July 24, 2008, DOI 10.1074/jbc.M805040200
Mei Du, Anu Rambhadran, and Vasanthi Jayaraman1 From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030 The apo state structure of the isolated ligand binding domain of the GluR6 subunit and the conformational changes induced by agonist binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 ligand binding domain, indicating that the cleft for the apo state of the GluR6 ligand binding domain should be similar to the UBP-310bound form of GluR5. This observation implies that the apo state of GluR6 undergoes a cleft closure of 29 –30° upon binding full agonists, one of the largest observed in the glutamate receptor family.
Ionotropic glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Glutamate binds to an extracellular domain in these receptors and mediates a series of conformational changes that ultimately result in the formation of a cation-selective channel (activation), which then subsequently closes in the continued presence of the agonist (desensitization) (1–7). The structures of the isolated ligand binding domain of the three subtypes of glutamate receptors have provided the first insight into the conformational changes and mechanism by which the agonist could control the activation and desensitization of the channel (8 –17). However, most of the structures are of the ␣-amino-5-
* This work was supported, in whole or in part, by National Institutes of Health Grant R01GM073102. This work was also supported by National Science Foundation Grant MCB-0444352. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 To whom correspondence should be addressed: MSB 4.106, 6431 Fannin St., Dept. of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, TX 77030. Tel.: 713-500-6236; Fax: 713-500-7444; E-mail: [email protected].
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methyl-3-hydroxy-4-isoxazole propionate (AMPA)2 subtype, for which currently there are over 60 structures in various ligated states (8, 15, 18). There is also significant insight into the dynamic state of the ligand binding domain for the AMPA receptors and on the role of specific agonist-protein interactions (3, 16, 19 –32). The kainate subtype, on the other hand, is the least studied of the three ionotropic glutamate receptor subtypes. The few structures of the agonist- and antagonistbound states of the ligand binding domains of the GluR6 and GluR5 subunits of kainate receptors suggest that this subtype of receptors most likely exhibits a similar mechanism as that of the closely related AMPA subtype (13, 14, 17, 18, 33), where the extent of activation is controlled by the extent of cleft closure induced by agonist binding in the ligand binding domain. However, the structures of the agonist-bound forms of kainate receptors cover a limited range of activations; the only partial agonist structures available are those of the kainate- and domoate-bound states, which exhibit 50% of the efficacy as the full agonist glutamate (13, 14, 18, 33). Additionally, there are no structures of the apo state for any subunit of the kainate receptors. Typically, the antagonist-bound structures are thought to closely resemble the apo state of the protein because they hold the binding domain in an open cleft conformation. In the case of kainate receptors, there are three antagonist-bound states of the GluR5 ligand binding domain. These three structures, however, show a range of cleft closures. Additionally, because of a shift in the orientation of domain 2, the axis of rotation between the antagonist- and glutamate-bound forms are different for the 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO)-bound state and the 2-amino-2-carboxyethyl-3-(2-carboxythiophene-3-ylmethyl)-5-methylpyrimidine-2,4-dione (UBP-310)- and (S)1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl) pyrimidine-2,4-dione (UBP-302)-bound states (18, 33). It is therefore still unknown which of these structures most closely represents the apo state of the protein.
2
The abbreviations used are: AMPA, ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate; ATPO, 2-amino-3-[5-tert-butyl-3-(phosphono-methoxy)-4-isoxazolyl] propionic acid; domoic acid, (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid; GluR5, glutamate receptor subunit 5; GluR6, glutamate receptor subunit 6; LRET, luminescence resonance energy transfer; TTHA, triethylenetetraaminehexaacetic acid chelate of terbium; UBP-302, (S)-1-(2-amino-2-carboxyethyl)3-(2-carboxybenzyl) pyrimidine-2,4-dione; UBP-310, 2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methyl pyrimidine-2,4-dione.
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Conformational Changes of Kainate Receptor
FIGURE 1. Structure of the ligand binding domain of GluR6 showing the sites labeled with donor and acceptor fluorophores. Because residue 398 is not observed in the crystal structure, the first residue 399 is highlighted.
To study the conformational changes in the isolated ligand binding domain of the kainate receptor, we have used a luminescence resonance energy (LRET)-based method to determine distances between sites on domain 1 and domain 2 of the bilobed structure of the GluR6 subunit of the kainate receptor (Fig. 1) in scenarios with a wide range of activations. The distance between the two domains provides a readout of the extent of cleft closure in the apo state and the various agonist-bound states. A wide range of activations was achieved by investigating a number of agonists with the wild type receptor and the T661E mutant. The T661E GluR6 receptors exhibit 20% of the maximum whole cell current by kainate relative to glutamate in the absence of desensitization (34). These LRET-based distances obtained for the wild type and T661E mutant provide a comprehensive analysis of the changes in cleft closure over the entire spectrum of activations and conclusively show that kainate receptors exhibit a graded cleft closure conformational change and hence exhibit a similar mechanism of activation as AMPA receptors.
EXPERIMENTAL PROCEDURES Chemicals—Domoic acid and (2S,4R)-4-methyl glutamate were purchased from Tocris Bioscience (Ellisville, MO). Glutamate and kainate were obtained from Sigma. [3H]Kainate was purchased from PerkinElmer Life Sciences. Purification of GluR6 Ligand Binding Domain—The GluR6flip plasmid was provided by Dr. Kathryn Partin (Colorado State University), and the GluR6 ligand binding domain plasmid was constructed as in previously described work (13). The mutations were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All constructs and mutations were confirmed by DNA sequencing. Proteins OCTOBER 3, 2008 • VOLUME 283 • NUMBER 40
were expressed, purified, and characterized as detailed previously (13). Briefly, the protein was expressed in Escherichia coli Origami-B (DE3) cells, and first purified by a nickel-nitrilotriacetic acid HiTrap affinity column (GE Healthcare) followed by thrombin digestion overnight to remove the histidine tag. Anion exchange chromatography (Q-Sepharose Fast Flow from GE Healthcare) was then performed to remove the thrombin. The functionality of all purified proteins was established by saturation binding of [3H]kainate to the proteins (supplemental Fig. 1). Labeling and LRET Measurements— 0.5 M protein in phosphate-buffered saline with 1 mM glutamate was labeled with a 1:1 ratio of the maleimide derivatives of fluorescein (Biotium, Hayward, CA) and triethylenetetraaminehexaacetic acid chelate of terbium (TTHA-Tb) (Invitrogen) or with terbium chelate alone for donor:acceptor and donor-only proteins, respectively. The labeled protein was dialyzed extensively against phosphate-buffered saline and used for the LRET measurements. Fluorescence measurements were obtained using a TimeMasterTM model TM-3/2003 (Photon Technology International, Inc., Birmingham, NJ) lifetime spectrometer. The fluorescence data were obtained with at least two different protein samples, and for each sample the data were an average of five shots (hence the data shown is an average of 10 data points). The data from each independent sample were also fit to ensure no significant differences between samples of the same protein. The distances were calculated from the donor-only and donor: acceptor lifetimes using Fo¨rster’s theory for energy transfer, and the R0 values were determined for each of the constructs by obtaining the fluorescence and absorption spectrum of donor and acceptor, respectively, tagged to the proteins (16). The error reported for the distances has been determined using error propagation due to the error in the lifetimes. Electrophysiological Measurements—For the whole cell current recordings, human embryonic kidney 293 cells were transfected with wild type or T661E mutant GluR6-flip receptors. The transfected cells were voltage-clamped at a holding potential of ⫺60 mV, and solutions were applied using a homemade U-tube mixing device that had a 100-m aperture. The electrode solution, for the electrophysiological measurements, contained 140 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM EGTA, 2 mM Na2ATP, and 10 mM HEPES (pH 7.4); the extracellular bath solution contained 145 mM NaCl, 1.8 mM MgCl2, 1 mM CaCl2, 3 mM KCl, 10 mM glucose, and 10 mM HEPES (pH 7.4). Currents were amplified with an Axon 200B amplifier (Molecular Devices, Sunnyvale, CA), low pass filtered at 1 kHz. The filtered signal was digitized using a Labmaster DMA digitizing board controlled by Axon PClamp software. All the experiments were performed at room temperature.
RESULTS AND DISCUSSION Conformational Changes Induced upon Ligand Binding— The LRET lifetimes as measured by the sensitized emission of the acceptor fluorescein due to luminescence transfer from the donor terbium chelate for the wild type and T661E mutant proteins are shown in Fig. 2 and listed in Table 1. There are currently two structures, the kainate-bound and glutamateJOURNAL OF BIOLOGICAL CHEMISTRY
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Conformational Changes of Kainate Receptor kainate, domoate, methyl glutamate, and glutamate (supplemental Fig. 2). The extent of activation was determined from the maximal whole cell currents mediated by saturating concentrations of the various agonists normalized to the currents mediated by saturating concentrations of glutamate. It should be noted that the normalized activation does not represent the channel open probability. Hence although the normalized activation is 1 for glutamate, the normalized open probability for glutamate activating the GluR6(Q)-flip receptors has been previously reported to be 0.96 (35). The correlation between the changes in the LRET-based distances between residue 398, in domain 1, and residues 666 and 689, in domain 2, for the different ligands bound to the wild type and T661E FIGURE 2. Fluorescence lifetimes. The LRET lifetime as measured by the sensitized emission of the acceptor in mutant and activation is shown in the GluR6 ligand binding domain labeled with fluorescein and TTHA-Tb in the apo (black) and kainate- (red), domoate- (blue), glutamate- (green), and methyl glutamate-bound (magenta) states for the wild type S398C- Fig. 3. These results show that the S666C (A) and wild type S398C-S689C (B); and in the apo (black), kainate- (red) and glutamate-bound (green) distances between residue 398 and states for S398C-S666C-T661E (C) and S398C-S689C-T661E (D). residues 666 and 689 exhibit a negative correlation to the extent of activation over the full range, i.e. the full agonists exhibit TABLE 1 shorter distances and partial agonists exhibit longer distances Lifetimes for donor-only (D) and donor:acceptor (DA)-tagged GluR6 ligand binding domain constructs and distances determined based between the sets of residues. Because residue 398 is in domain 1 on these lifetimes and residues 666 and 689 are in domain 2 of the isolated ligand Protein Ligated state D DA Distance binding domain, the decrease in distance between the two s s Å domains is consistent with a cleft closure conformational S398C-S666C Apo 1699 ⫾ 27 192 ⫾ 2 42.5 ⫾ 0.7 change. Thus, it can be concluded that agonists with greater Glutamate 1745 ⫾ 32 130 ⫾ 3 39.4 ⫾ 0.9 Kainate 1753 ⫾ 34 149 ⫾ 4 40.3 ⫾ 1 activations induce larger cleft closure, and cleft closure could be Domoate 1712 ⫾ 30 144 ⫾ 3 40.3 ⫾ 1 the coupling mechanism by which the agonist mediates chanMethyl glutamate 1675 ⫾ 27 129 ⫾ 3 39.6 ⫾ 0.9 S398C-S689C Apo 1745 ⫾ 24 185 ⫾ 2 42.0 ⫾ 0.6 nel opening over the full range of activations. Glutamate 1774 ⫾ 12 84 ⫾ 2 36.3 ⫾ 0.9 Apo State of the Protein—The LRET-based distances indicate Kainate 1718 ⫾ 23 97 ⫾ 2 37.5 ⫾ 0.8 Domoate 1684 ⫾ 24 96 ⫾ 2 37.5 ⫾ 0.8 that the apo state is more open relative to the agonist-bound Methyl glutamate 1721 ⫾ 22 75 ⫾ 2 35.8 ⫾ 1 states, with a 3.1- and 5.7-Å increase in distance between resiS398C-S666C-T661E Apo 1786 ⫾ 24 212 ⫾ 3 42.9 ⫾ 0.6 Glutamate 1733 ⫾ 32 135 ⫾ 3 39.7 ⫾ 0.9 due 398 and residues 666 and 689, respectively, upon going Kainate 1777 ⫾ 25 174 ⫾ 3 41.4 ⫾ 0.6 from the glutamate-bound form to the apo state. This result is S398C-S689C-T661E Apo 1711 ⫾ 30 176 ⫾ 2 41.8 ⫾ 0.7 Glutamate 1698 ⫾ 34 66 ⫾ 1 35.1 ⫾ 0.7 consistent with a cleft closure conformational change induced Kainate 1690 ⫾ 31 98 ⫾ 2 37.7 ⫾ 0.8 by agonist binding. Because there are no apo state or antagonist-bound strucbound forms, available for the GluR6 ligand binding domain tures available for the GluR6 ligand binding domain, the dis(13). The changes in distance calculated from the LRET life- tance change between the apo state and glutamate-bound state times between the kainate- and glutamate-bound states for res- for GluR6 is compared with the changes between the three idues 398 – 666 and 398 – 689 are 1 and 1.5 Å, respectively, for antagonist-bound structures and the glutamate-bound strucboth sets of residues, similar to the changes of 0.9 and 1.2 Å ture for the closely related ligand binding domain of the GluR5 observed between the kainate and glutamate crystal structures subunit (18, 33). It should be noted that the glutamate-bound (13). This agreement in distance changes establishes that these forms of the GluR6 and GluR5 ligand binding domains exhibit two x-ray structures are a good representation of the average similar distances between residue 398 and residues 666 and 689, thus allowing for such a comparison (18, 33). dynamic structure of the protein in buffer. The three antagonist-bound structures of GluR5 have To obtain a comprehensive correlation between the extent of cleft closure and activation, a wide range of activations was slightly varying degrees of open clefts relative to the full agonist investigated using the wild type and T661E mutant and agonists glutamate-bound state, with the cleft being 28 –30° more open
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Conformational Changes of Kainate Receptor similar to that observed in the UBP310- and UBP-302-bound forms and not similar to that observed for the ATPO-bound state of GluR5 ligand binding domain, and therefore the apo state of the GluR6 ligand binding domain should be similar in structure to the UBP-310and UBP-302-bound structures of the GluR5 ligand binding domain. The similarity of the distances in the apo state with UBP-310- and UBPFIGURE 3. Dependence of cleft closure versus extent of activation. LRET-based distances for the S398C- 302-bound structures also implies S666C wild type (filled square) and S398C-S666C-T661E (open square) (A) S398C-S689C wild type (filled square) and S398C-S689C-T661E (open square) (B) GluR6 ligand binding domain labeled with fluorescein and terbium that the cleft is as open as in the chelate, plotted as a function of extent of activation of the receptor as determined by the maximum currents at UBP-310- and UBP-302-bound saturating concentrations of agonist normalized to the currents mediated by saturating concentrations of structures and that the change in glutamate. cleft closure upon binding agonists in GluR6 would be ⬃29 –30°, which is one of the largest observed in this class of proteins. Although the LRET investigations presented here probe a large range of activations, they are still not as comprehensive as that for the AMPA subtype. A detailed study such as the one performed for the GluR2 subunit (16) might reveal mutants and ligands that may not follow the general correlations FIGURE 4. Overlaid structures of GluR5 ligand binding domain in the ATPO- (red) and glutamate-bound between the extent of cleft closure (blue) forms (A), UBP-310- (red) and glutamate-bound (blue) forms (B); UBP-302- (red) and glutamateand the extent of activation. Hence, bound (blue) forms (C). The axis of rotation for the cleft closure conformational change between the two although the cleft closure correlaoverlaid forms is shown as a dashed line. tion holds for a number of cases, it in the ATPO, UBP-310, and UBP-302 antagonist-bound states should be noted that this may not be the only means by which (18, 33). Apart from the small changes in the extent of cleft the agonist controls receptor activation. A more detailed study opening, the axis of rotation in the ATPO structure is also dif- of the dynamics of the GluR6 subunit by other spectroscopic ferent from that in the UBP-310- and UBP-302-bound states investigations such as NMR and vibrational spectroscopy (Fig. 4). This difference in the axis of rotation is evident in the would provide more detailed insight into the various different distances between 398 and 666 and 398 and 689 in the GluR5 mechanisms by which the agonists mediate receptor activation crystal structures; in the ATPO-bound state, these differences and provide insight into further similarities or differences in distances between the antagonist-bound and glutamate- between AMPA and kainate receptors. bound states are not significantly different between the two sets Conclusions—The LRET investigations of the kainate subof residues, 5.3 and 6.7 Å, whereas in the UBP-310- and UBP- type show that, as with AMPA receptors, the extent of cleft 302-bound states the differences in distances are significantly closure is graded and correlates to the extent of activation different, 3.8 and 6.7 Å and 3.9 and 7.1 Å, respectively. The 3.1- and suggest that the mechanism of activation of kainate and 5.7-Å distance changes between the apo and glutamate- receptors is similar to AMPA receptors. However, the LRET bound states for the two sets of residues from the GluR6 LRET investigations in conjunction with the crystal structure studmeasurements are similar to the 3.8- and 6.7-Å distance ies (18, 33) suggest that the change in degree of cleft closure changes observed in the GluR5 crystal structures for the UBP- in kainate receptors from the apo state to the full agonist 310-bound state and are also not significantly different from the glutamate-bound states is much greater than in AMPA 3.9- and 7.1-Å distance changes observed in the UBP-302- receptors, suggesting that although the overall mechanism bound state. The 3.1-Å distance change from residue 398 – 666 of activation (cleft closure of the ligand binding domain) may determined from the GluR6 LRET measurements of the apo be the same, the subtleties in mechanism between the two state to the glutamate-bound state, however, is significantly dif- subtypes of receptors may be different. ferent from the 5.3-Å distance change in the ATPO-bound state to the glutamate-bound state observed in the GluR5 crys- Acknowledgment—We thank Kimberly Mankiewicz for editorial tal structures. These results suggest that the axis for rotation in help. the apo state of the GluR6 ligand binding domain should be OCTOBER 3, 2008 • VOLUME 283 • NUMBER 40
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Conformational Changes of Kainate Receptor REFERENCES 1. Dingledine, R., Borges, K., Bowie, D., and Traynelis, S. F. (1999) Pharmacol. Rev. 51, 7– 61 2. Madden, D. R. (2002) Nat. Rev. Neurosci. 3, 91–101 3. McFeeters, R. L., and Oswald, R. E. (2004) FASEB J. 18, 428 – 438 4. Oswald, R. E. (2004) Adv. Protein Chem. 68, 313–349 5. Mayer, M. L. (2006) Nature 440, 456 – 462 6. Gouaux, E. (2004) J. Physiol. (Lond.) 554, 249 –253 7. Mayer, M. L. (2005) Curr. Opin. Neurobiol. 15, 282–288 8. Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165–181 9. Furukawa, H., and Gouaux, E. (2003) EMBO J. 22, 2873–2885 10. Furukawa, H., Singh, S. K., Mancusso, R., and Gouaux, E. (2005) Nature 438, 185–192 11. Armstrong, N., Jasti, J., Beich-Frandsen, M., and Gouaux, E. (2006) Cell 127, 85–97 12. Sun, Y., Olson, R., Horning, M., Armstrong, N., Mayer, M., and Gouaux, E. (2002) Nature 417, 245–253 13. Mayer, M. L. (2005) Neuron 45, 539 –552 14. Nanao, M. H., Green, T., Stern-Bach, Y., Heinemann, S. F., and Choe, S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1708 –1713 15. Mayer, M. L., and Armstrong, N. (2004) Annu. Rev. Physiol. 66, 161–181 16. Ramanoudjame, G., Du, M., Mankiewicz, K. A., and Jayaraman, V. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10473–10478 17. Naur, P., Vestergaard, B., Skov, L. K., Egebjerg, J., Gajhede, M., and Kastrup, J. S. (2005) FEBS Lett. 579, 1154 –1160 18. Mayer, M. L., Ghosal, A., Dolman, N. P., and Jane, D. E. (2006) J. Neurosci. 26, 2852–2861 19. Jayaraman, V., Keesey, R., and Madden, D. R. (2000) Biochemistry 39, 8693– 8697
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20. Madden, D. R., Thiran, S., Zimmermann, H., Romm, J., and Jayaraman, V. (2001) J. Biol. Chem. 276, 37821–37826 21. Cheng, Q., Thiran, S., Yernool, D., Gouaux, E., and Jayaraman, V. (2002) Biochemistry 41, 1602–1608 22. Deming, D., Cheng, Q., and Jayaraman, V. (2003) J. Biol. Chem. 278, 17589 –17592 23. Madden, D. R., Cheng, Q., Thiran, S., Rajan, S., Rigo, F., Keinanen, K., Reinelt, S., Zimmermann, H., and Jayaraman, V. (2004) Biochemistry 43, 15838 –15844 24. Jayaraman, V. (2004) Methods Enzymol. 380, 170 –187 25. Cheng, Q., and Jayaraman, V. (2004) J. Biol. Chem. 279, 26346 –26350 26. Cheng, Q., Du, M., Ramanoudjame, G., and Jayaraman, V. (2005) Nat. Chem. Biol. 1, 329 –332 27. Du, M., Reid, S. A., and Jayaraman, V. (2005) J. Biol. Chem. 280, 8633– 8636 28. Ahmed, A. H., Loh, A. P., Jane, D. E., and Oswald, R. E. (2007) J. Biol. Chem. 282, 12773–12784 29. Mankiewicz, K. A., and Jayaraman, V. (2007) Braz. J. Med. Biol. Res. 40, 1419 –1427 30. Mankiewicz, K. A., Rambhadran, A., Du, M., Ramanoudjame, G., and Jayaraman, V. (2007) Biochemistry 46, 1343–1349 31. Mankiewicz, K. A., Rambhadran, A., Wathen, L., and Jayaraman, V. (2008) Biochemistry 47, 398 – 404 32. Fenwick, M. K., and Oswald, R. E. (2008) J. Mol. Biol. 378, 673– 685 33. Hald, H., Naur, P., Pickering, D. S., Sprogoe, D., Madsen, U., Timmermann, D. B., Ahring, P. K., Liljefors, T., Schousboe, A., Egebjerg, J., Gajhede, M., and Kastrup, J. S. (2007) J. Biol. Chem. 282, 25726 –25736 34. Fleck, M. W., Cornell, E., and Mah, S. J. (2003) J. Neurosci. 23, 1219 –1227 35. Li, G., Oswald, R. E., and Niu, L. (2003) Biochemistry 42, 12367–12375
VOLUME 283 • NUMBER 40 • OCTOBER 3, 2008
Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a Kainate Receptor*□ S
Received for publication, July 2, 2008, and in revised form, July 23, 2008 Published, JBC Papers in Press, July 24, 2008, DOI 10.1074/jbc.M805040200
Mei Du, Anu Rambhadran, and Vasanthi Jayaraman1 From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030 The apo state structure of the isolated ligand binding domain of the GluR6 subunit and the conformational changes induced by agonist binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 ligand binding domain, indicating that the cleft for the apo state of the GluR6 ligand binding domain should be similar to the UBP-310bound form of GluR5. This observation implies that the apo state of GluR6 undergoes a cleft closure of 29 –30° upon binding full agonists, one of the largest observed in the glutamate receptor family.
Ionotropic glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Glutamate binds to an extracellular domain in these receptors and mediates a series of conformational changes that ultimately result in the formation of a cation-selective channel (activation), which then subsequently closes in the continued presence of the agonist (desensitization) (1–7). The structures of the isolated ligand binding domain of the three subtypes of glutamate receptors have provided the first insight into the conformational changes and mechanism by which the agonist could control the activation and desensitization of the channel (8 –17). However, most of the structures are of the ␣-amino-5-
* This work was supported, in whole or in part, by National Institutes of Health Grant R01GM073102. This work was also supported by National Science Foundation Grant MCB-0444352. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 To whom correspondence should be addressed: MSB 4.106, 6431 Fannin St., Dept. of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, TX 77030. Tel.: 713-500-6236; Fax: 713-500-7444; E-mail: [email protected].
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methyl-3-hydroxy-4-isoxazole propionate (AMPA)2 subtype, for which currently there are over 60 structures in various ligated states (8, 15, 18). There is also significant insight into the dynamic state of the ligand binding domain for the AMPA receptors and on the role of specific agonist-protein interactions (3, 16, 19 –32). The kainate subtype, on the other hand, is the least studied of the three ionotropic glutamate receptor subtypes. The few structures of the agonist- and antagonistbound states of the ligand binding domains of the GluR6 and GluR5 subunits of kainate receptors suggest that this subtype of receptors most likely exhibits a similar mechanism as that of the closely related AMPA subtype (13, 14, 17, 18, 33), where the extent of activation is controlled by the extent of cleft closure induced by agonist binding in the ligand binding domain. However, the structures of the agonist-bound forms of kainate receptors cover a limited range of activations; the only partial agonist structures available are those of the kainate- and domoate-bound states, which exhibit 50% of the efficacy as the full agonist glutamate (13, 14, 18, 33). Additionally, there are no structures of the apo state for any subunit of the kainate receptors. Typically, the antagonist-bound structures are thought to closely resemble the apo state of the protein because they hold the binding domain in an open cleft conformation. In the case of kainate receptors, there are three antagonist-bound states of the GluR5 ligand binding domain. These three structures, however, show a range of cleft closures. Additionally, because of a shift in the orientation of domain 2, the axis of rotation between the antagonist- and glutamate-bound forms are different for the 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO)-bound state and the 2-amino-2-carboxyethyl-3-(2-carboxythiophene-3-ylmethyl)-5-methylpyrimidine-2,4-dione (UBP-310)- and (S)1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl) pyrimidine-2,4-dione (UBP-302)-bound states (18, 33). It is therefore still unknown which of these structures most closely represents the apo state of the protein.
2
The abbreviations used are: AMPA, ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate; ATPO, 2-amino-3-[5-tert-butyl-3-(phosphono-methoxy)-4-isoxazolyl] propionic acid; domoic acid, (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid; GluR5, glutamate receptor subunit 5; GluR6, glutamate receptor subunit 6; LRET, luminescence resonance energy transfer; TTHA, triethylenetetraaminehexaacetic acid chelate of terbium; UBP-302, (S)-1-(2-amino-2-carboxyethyl)3-(2-carboxybenzyl) pyrimidine-2,4-dione; UBP-310, 2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methyl pyrimidine-2,4-dione.
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Conformational Changes of Kainate Receptor
FIGURE 1. Structure of the ligand binding domain of GluR6 showing the sites labeled with donor and acceptor fluorophores. Because residue 398 is not observed in the crystal structure, the first residue 399 is highlighted.
To study the conformational changes in the isolated ligand binding domain of the kainate receptor, we have used a luminescence resonance energy (LRET)-based method to determine distances between sites on domain 1 and domain 2 of the bilobed structure of the GluR6 subunit of the kainate receptor (Fig. 1) in scenarios with a wide range of activations. The distance between the two domains provides a readout of the extent of cleft closure in the apo state and the various agonist-bound states. A wide range of activations was achieved by investigating a number of agonists with the wild type receptor and the T661E mutant. The T661E GluR6 receptors exhibit 20% of the maximum whole cell current by kainate relative to glutamate in the absence of desensitization (34). These LRET-based distances obtained for the wild type and T661E mutant provide a comprehensive analysis of the changes in cleft closure over the entire spectrum of activations and conclusively show that kainate receptors exhibit a graded cleft closure conformational change and hence exhibit a similar mechanism of activation as AMPA receptors.
EXPERIMENTAL PROCEDURES Chemicals—Domoic acid and (2S,4R)-4-methyl glutamate were purchased from Tocris Bioscience (Ellisville, MO). Glutamate and kainate were obtained from Sigma. [3H]Kainate was purchased from PerkinElmer Life Sciences. Purification of GluR6 Ligand Binding Domain—The GluR6flip plasmid was provided by Dr. Kathryn Partin (Colorado State University), and the GluR6 ligand binding domain plasmid was constructed as in previously described work (13). The mutations were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All constructs and mutations were confirmed by DNA sequencing. Proteins OCTOBER 3, 2008 • VOLUME 283 • NUMBER 40
were expressed, purified, and characterized as detailed previously (13). Briefly, the protein was expressed in Escherichia coli Origami-B (DE3) cells, and first purified by a nickel-nitrilotriacetic acid HiTrap affinity column (GE Healthcare) followed by thrombin digestion overnight to remove the histidine tag. Anion exchange chromatography (Q-Sepharose Fast Flow from GE Healthcare) was then performed to remove the thrombin. The functionality of all purified proteins was established by saturation binding of [3H]kainate to the proteins (supplemental Fig. 1). Labeling and LRET Measurements— 0.5 M protein in phosphate-buffered saline with 1 mM glutamate was labeled with a 1:1 ratio of the maleimide derivatives of fluorescein (Biotium, Hayward, CA) and triethylenetetraaminehexaacetic acid chelate of terbium (TTHA-Tb) (Invitrogen) or with terbium chelate alone for donor:acceptor and donor-only proteins, respectively. The labeled protein was dialyzed extensively against phosphate-buffered saline and used for the LRET measurements. Fluorescence measurements were obtained using a TimeMasterTM model TM-3/2003 (Photon Technology International, Inc., Birmingham, NJ) lifetime spectrometer. The fluorescence data were obtained with at least two different protein samples, and for each sample the data were an average of five shots (hence the data shown is an average of 10 data points). The data from each independent sample were also fit to ensure no significant differences between samples of the same protein. The distances were calculated from the donor-only and donor: acceptor lifetimes using Fo¨rster’s theory for energy transfer, and the R0 values were determined for each of the constructs by obtaining the fluorescence and absorption spectrum of donor and acceptor, respectively, tagged to the proteins (16). The error reported for the distances has been determined using error propagation due to the error in the lifetimes. Electrophysiological Measurements—For the whole cell current recordings, human embryonic kidney 293 cells were transfected with wild type or T661E mutant GluR6-flip receptors. The transfected cells were voltage-clamped at a holding potential of ⫺60 mV, and solutions were applied using a homemade U-tube mixing device that had a 100-m aperture. The electrode solution, for the electrophysiological measurements, contained 140 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM EGTA, 2 mM Na2ATP, and 10 mM HEPES (pH 7.4); the extracellular bath solution contained 145 mM NaCl, 1.8 mM MgCl2, 1 mM CaCl2, 3 mM KCl, 10 mM glucose, and 10 mM HEPES (pH 7.4). Currents were amplified with an Axon 200B amplifier (Molecular Devices, Sunnyvale, CA), low pass filtered at 1 kHz. The filtered signal was digitized using a Labmaster DMA digitizing board controlled by Axon PClamp software. All the experiments were performed at room temperature.
RESULTS AND DISCUSSION Conformational Changes Induced upon Ligand Binding— The LRET lifetimes as measured by the sensitized emission of the acceptor fluorescein due to luminescence transfer from the donor terbium chelate for the wild type and T661E mutant proteins are shown in Fig. 2 and listed in Table 1. There are currently two structures, the kainate-bound and glutamateJOURNAL OF BIOLOGICAL CHEMISTRY
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Conformational Changes of Kainate Receptor kainate, domoate, methyl glutamate, and glutamate (supplemental Fig. 2). The extent of activation was determined from the maximal whole cell currents mediated by saturating concentrations of the various agonists normalized to the currents mediated by saturating concentrations of glutamate. It should be noted that the normalized activation does not represent the channel open probability. Hence although the normalized activation is 1 for glutamate, the normalized open probability for glutamate activating the GluR6(Q)-flip receptors has been previously reported to be 0.96 (35). The correlation between the changes in the LRET-based distances between residue 398, in domain 1, and residues 666 and 689, in domain 2, for the different ligands bound to the wild type and T661E FIGURE 2. Fluorescence lifetimes. The LRET lifetime as measured by the sensitized emission of the acceptor in mutant and activation is shown in the GluR6 ligand binding domain labeled with fluorescein and TTHA-Tb in the apo (black) and kainate- (red), domoate- (blue), glutamate- (green), and methyl glutamate-bound (magenta) states for the wild type S398C- Fig. 3. These results show that the S666C (A) and wild type S398C-S689C (B); and in the apo (black), kainate- (red) and glutamate-bound (green) distances between residue 398 and states for S398C-S666C-T661E (C) and S398C-S689C-T661E (D). residues 666 and 689 exhibit a negative correlation to the extent of activation over the full range, i.e. the full agonists exhibit TABLE 1 shorter distances and partial agonists exhibit longer distances Lifetimes for donor-only (D) and donor:acceptor (DA)-tagged GluR6 ligand binding domain constructs and distances determined based between the sets of residues. Because residue 398 is in domain 1 on these lifetimes and residues 666 and 689 are in domain 2 of the isolated ligand Protein Ligated state D DA Distance binding domain, the decrease in distance between the two s s Å domains is consistent with a cleft closure conformational S398C-S666C Apo 1699 ⫾ 27 192 ⫾ 2 42.5 ⫾ 0.7 change. Thus, it can be concluded that agonists with greater Glutamate 1745 ⫾ 32 130 ⫾ 3 39.4 ⫾ 0.9 Kainate 1753 ⫾ 34 149 ⫾ 4 40.3 ⫾ 1 activations induce larger cleft closure, and cleft closure could be Domoate 1712 ⫾ 30 144 ⫾ 3 40.3 ⫾ 1 the coupling mechanism by which the agonist mediates chanMethyl glutamate 1675 ⫾ 27 129 ⫾ 3 39.6 ⫾ 0.9 S398C-S689C Apo 1745 ⫾ 24 185 ⫾ 2 42.0 ⫾ 0.6 nel opening over the full range of activations. Glutamate 1774 ⫾ 12 84 ⫾ 2 36.3 ⫾ 0.9 Apo State of the Protein—The LRET-based distances indicate Kainate 1718 ⫾ 23 97 ⫾ 2 37.5 ⫾ 0.8 Domoate 1684 ⫾ 24 96 ⫾ 2 37.5 ⫾ 0.8 that the apo state is more open relative to the agonist-bound Methyl glutamate 1721 ⫾ 22 75 ⫾ 2 35.8 ⫾ 1 states, with a 3.1- and 5.7-Å increase in distance between resiS398C-S666C-T661E Apo 1786 ⫾ 24 212 ⫾ 3 42.9 ⫾ 0.6 Glutamate 1733 ⫾ 32 135 ⫾ 3 39.7 ⫾ 0.9 due 398 and residues 666 and 689, respectively, upon going Kainate 1777 ⫾ 25 174 ⫾ 3 41.4 ⫾ 0.6 from the glutamate-bound form to the apo state. This result is S398C-S689C-T661E Apo 1711 ⫾ 30 176 ⫾ 2 41.8 ⫾ 0.7 Glutamate 1698 ⫾ 34 66 ⫾ 1 35.1 ⫾ 0.7 consistent with a cleft closure conformational change induced Kainate 1690 ⫾ 31 98 ⫾ 2 37.7 ⫾ 0.8 by agonist binding. Because there are no apo state or antagonist-bound strucbound forms, available for the GluR6 ligand binding domain tures available for the GluR6 ligand binding domain, the dis(13). The changes in distance calculated from the LRET life- tance change between the apo state and glutamate-bound state times between the kainate- and glutamate-bound states for res- for GluR6 is compared with the changes between the three idues 398 – 666 and 398 – 689 are 1 and 1.5 Å, respectively, for antagonist-bound structures and the glutamate-bound strucboth sets of residues, similar to the changes of 0.9 and 1.2 Å ture for the closely related ligand binding domain of the GluR5 observed between the kainate and glutamate crystal structures subunit (18, 33). It should be noted that the glutamate-bound (13). This agreement in distance changes establishes that these forms of the GluR6 and GluR5 ligand binding domains exhibit two x-ray structures are a good representation of the average similar distances between residue 398 and residues 666 and 689, thus allowing for such a comparison (18, 33). dynamic structure of the protein in buffer. The three antagonist-bound structures of GluR5 have To obtain a comprehensive correlation between the extent of cleft closure and activation, a wide range of activations was slightly varying degrees of open clefts relative to the full agonist investigated using the wild type and T661E mutant and agonists glutamate-bound state, with the cleft being 28 –30° more open
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Conformational Changes of Kainate Receptor similar to that observed in the UBP310- and UBP-302-bound forms and not similar to that observed for the ATPO-bound state of GluR5 ligand binding domain, and therefore the apo state of the GluR6 ligand binding domain should be similar in structure to the UBP-310and UBP-302-bound structures of the GluR5 ligand binding domain. The similarity of the distances in the apo state with UBP-310- and UBPFIGURE 3. Dependence of cleft closure versus extent of activation. LRET-based distances for the S398C- 302-bound structures also implies S666C wild type (filled square) and S398C-S666C-T661E (open square) (A) S398C-S689C wild type (filled square) and S398C-S689C-T661E (open square) (B) GluR6 ligand binding domain labeled with fluorescein and terbium that the cleft is as open as in the chelate, plotted as a function of extent of activation of the receptor as determined by the maximum currents at UBP-310- and UBP-302-bound saturating concentrations of agonist normalized to the currents mediated by saturating concentrations of structures and that the change in glutamate. cleft closure upon binding agonists in GluR6 would be ⬃29 –30°, which is one of the largest observed in this class of proteins. Although the LRET investigations presented here probe a large range of activations, they are still not as comprehensive as that for the AMPA subtype. A detailed study such as the one performed for the GluR2 subunit (16) might reveal mutants and ligands that may not follow the general correlations FIGURE 4. Overlaid structures of GluR5 ligand binding domain in the ATPO- (red) and glutamate-bound between the extent of cleft closure (blue) forms (A), UBP-310- (red) and glutamate-bound (blue) forms (B); UBP-302- (red) and glutamateand the extent of activation. Hence, bound (blue) forms (C). The axis of rotation for the cleft closure conformational change between the two although the cleft closure correlaoverlaid forms is shown as a dashed line. tion holds for a number of cases, it in the ATPO, UBP-310, and UBP-302 antagonist-bound states should be noted that this may not be the only means by which (18, 33). Apart from the small changes in the extent of cleft the agonist controls receptor activation. A more detailed study opening, the axis of rotation in the ATPO structure is also dif- of the dynamics of the GluR6 subunit by other spectroscopic ferent from that in the UBP-310- and UBP-302-bound states investigations such as NMR and vibrational spectroscopy (Fig. 4). This difference in the axis of rotation is evident in the would provide more detailed insight into the various different distances between 398 and 666 and 398 and 689 in the GluR5 mechanisms by which the agonists mediate receptor activation crystal structures; in the ATPO-bound state, these differences and provide insight into further similarities or differences in distances between the antagonist-bound and glutamate- between AMPA and kainate receptors. bound states are not significantly different between the two sets Conclusions—The LRET investigations of the kainate subof residues, 5.3 and 6.7 Å, whereas in the UBP-310- and UBP- type show that, as with AMPA receptors, the extent of cleft 302-bound states the differences in distances are significantly closure is graded and correlates to the extent of activation different, 3.8 and 6.7 Å and 3.9 and 7.1 Å, respectively. The 3.1- and suggest that the mechanism of activation of kainate and 5.7-Å distance changes between the apo and glutamate- receptors is similar to AMPA receptors. However, the LRET bound states for the two sets of residues from the GluR6 LRET investigations in conjunction with the crystal structure studmeasurements are similar to the 3.8- and 6.7-Å distance ies (18, 33) suggest that the change in degree of cleft closure changes observed in the GluR5 crystal structures for the UBP- in kainate receptors from the apo state to the full agonist 310-bound state and are also not significantly different from the glutamate-bound states is much greater than in AMPA 3.9- and 7.1-Å distance changes observed in the UBP-302- receptors, suggesting that although the overall mechanism bound state. The 3.1-Å distance change from residue 398 – 666 of activation (cleft closure of the ligand binding domain) may determined from the GluR6 LRET measurements of the apo be the same, the subtleties in mechanism between the two state to the glutamate-bound state, however, is significantly dif- subtypes of receptors may be different. ferent from the 5.3-Å distance change in the ATPO-bound state to the glutamate-bound state observed in the GluR5 crys- Acknowledgment—We thank Kimberly Mankiewicz for editorial tal structures. These results suggest that the axis for rotation in help. the apo state of the GluR6 ligand binding domain should be OCTOBER 3, 2008 • VOLUME 283 • NUMBER 40
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