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The EMBO Journal Review Process File - EMBO-2009-71022 Manuscript EMBO-2009-71022 Crystal structure and association behavior of the GluR2 amino terminal domain Rongsheng Jin, Satinder K. Singh, Shenyan Gu, Hiroyasu Furukawa, Alexander Sobolevsky, Jie Zhou, Yan Jin Corresponding author: Eric Gouaux, Oregon Health & Science University / Howard Hughes Medical Institute Review timeline: Submission date: Editorial Decision: Revision received: Accepted: 03 April 2009 22 April 2009 26 April 2009 27 April 2009 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 22 April 2009 I have received the third report on your paper and thank you first of all for submitting your research manuscript for consideration to The EMBO Journal editorial office. From the enclosed comments you will see, that all three referee's essentially appreciate the high technical quality of this long awaited structure. Ref#2 has a few remarks related to caution some of the interpretations, at least in the light of no further experimental validation being provided. I believe that some of his/her concerns could be addressed by more strongly referring to available mutational and pharmacological data, as indicated by ref#1. Similarly, ref#3 recommends a few minor changes, to more adequately express what can be really concluded from the experimental evidence. These overall very positive conclusions seem to enable re-submission of a revised version without further experimentation, still carefully addressing all the concerns raised from our referee's. I do have to remind you though that it is EMBO_J policy to allow a single round of revisions only, which means that the final decision on acceptance or rejection depends entirely on the content of the ultimate version of your manuscript. Thank you for the opportunity to consider your work for publication. I look forward to reading the revised manuscript. REFEREE REPORTS Referee #1 (Remarks to the Author): This report describes the resolved structure of the amino terminal domain of the GluR2 AMPA © European Molecular Biology Organization 1 The EMBO Journal Review Process File - EMBO-2009-71022 receptor. It represents the first such description and provides unparalleled insight into the physical nature of these domains in glutamate receptors. Analysis of the structure provides insight into previous results from mutagenic and pharmacological studies. The technical quality of the study is excellent, as is typical from this research group, and the manuscript is very well-written. I have no criticisms of this high-quality, high-impact research study. Referee #2 (Remarks to the Author): This manuscript addresses an important gap in our understanding of glutamate receptor structure, and in this way it is an important addition to the literature. There are a number of issues that could be considered in an effort to improve the manuscript. Major comment: 1. There is no functional experimentation to show that the dimer interface observed is relevant or correctly described for full-length receptors. Whereas one might be tempted to argue that the dimer interface as defined by crystallography is almost certainly correct, one need only revisit 2003 when this same group reported the dimer interface between two NR1 glycine binding domains. Some months later, the same group reported that the NMDA ligand binding domain dimer was made up of NR1/NR2 (not NR1/NR1). Whereas it may turn out that for some NMDA receptors NR1/NR1 ligand binding dimers are relevant, a bit of caution still might be warranted here. 2. Overall, the manuscript discusses exciting data describing the crystal structure of the GluR2ATD. The structure is described in detail and structural features of the GluR2-ATD are compared to expected structures of the ATDs of kainate and NMDA receptors based on sequence alignments. However, when the GluR2-ATD structure is used to speculate on the function of the NMDA receptor ATDs and their ligand binding sites, the comparison and the extrapolation of the findings become tenuous. Based on homology models of the ATDs and extensive functional data, several papers from multiple investigators have (already) suggested that Zn2+ and ifenprodil bind in a crevice formed between two lobes in the ATD. The authors did not build homology models of the NR2A-ATD and the NR2B-ATD before mapping the residues important for ligand binding in Figure 8 and consequently, Figure 8 and the discussion concerning this figure do not add important new information to characteristics of the Zn2+ and ifenprodil binding sites that isn't already in the literature. The authors should stick to the data that is included in the paper, which are the crystal structures and the sedimentation equilibrium studies; removal of figure 8 and table 3 would only strengthen the paper by focusing the text on the data at hand. 3. Following on this theme, the notion that Zn2+ and ifenprodil share a common mechanism of modulation that involves closure of the ATD has been proposed before (see for example Gielen etal., Neuron, 2008). Little is added here to what has been shown in that study. 4. The discussion concerning the mechanism by which spermine acts to potentiate NMDA receptors also seems premature. Wouldn't it be better left to future studies to explain this mechanism - perhaps using the GluR2-ATD structure to guide functional experimentation? 5. The authors should also reduce the section on the location of the modulatory bindings sites in NMDA receptors and make sure to cite the appropriate papers that have studied the structural features of Zn2+ and ifenprodil inhibition when they are mentioned. The authors should also cite the papers that have shown that AMPA receptor ATDs are not important for function (for example Pasternack et al., JBC, 2002). 6. The conclusion should be revised. The present study does not "demonstrate the roles that residues, located at the dimer interface, play in defining receptor subtype specific assembly". Rather, the study elaborates on one mechanism by which receptor subtype specific assembly is achieved for the GluR2 AMPA receptor. Demonstrating this would require mutagenesis and experimental data on the assembly of full-length receptors. Furthermore, the sentences on tetramer formation seem to be speculation rather than conclusion. The conclusion should be revised, and the last sentence of the abstract also revised. © European Molecular Biology Organization 2 The EMBO Journal Review Process File - EMBO-2009-71022 Minor comments: 6. Page 5, line 11: "GluRD" should be "GluR4" in order to be use a consistent nomenclature throughout the manuscript. 7. Page 6, give the residues comprising the construct in the main text. 8. Page 9, line 17: "strand-d" should be "beta4" in order to be use a consistent nomenclature throughout the manuscript. 9. Page 14, line 8: "R1 domain" should be "L1 domain". Referee #3 (Remarks to the Author): This is a report of the long-awaited structure of the GluR2 ATD. It provides a new breakthrough in the field and will be of considerable interest to the general readership of the EMBO Journal. I have only a few minor suggestions: 1) On page 9, the paragraph comparing the ATD structure with the sequences of AMPA, kainate and NMDA receptors seems out of place. 2) On page 10, I think it is an overinterpretation of the crystal data to suggest that unliganded protein cannot adopt multiple states. All that can be said is that under the conditions that the protein crystallized, only one conformation is seen. 3) On page 19, the authors conclude that tetramerization must derive from the transmembrane domains. I think this also goes beyond what can be reasonably concluded from the data. One can easily envision a model in which very low affinity interactions between ATD dimers, LBD dimers and the transmembrane domains can sum (that is, the sum of the free energies), making a very strong overall interaction. So, whereas their statement may be true, this conclusion cannot be directly made from the data presented in the paper. 1st Revision - authors' response 26 April 2009 Referee #1 (Remarks to the Author): This report describes the resolved structure of the amino terminal domain of the GluR2 AMPA receptor. It represents the first such description and provides unparalleled insight into the physical nature of these domains in glutamate receptors. Analysis of the structure provides insight into previous results from mutagenic and pharmacological studies. The technical quality of the study is excellent, as is typical from this research group, and the manuscript is very well-written. I have no criticisms of this high-quality, high-impact research study. Reply: We are heartened by this enthusiastic response and believe that the changes we have made (see below) further strengthen an already strong manuscript. Referee #2 (Remarks to the Author): This manuscript addresses an important gap in our understanding of glutamate receptor structure, and in this way it is an important addition to the literature. There are a number of issues that could be considered in an effort to improve the manuscript. © European Molecular Biology Organization 3 The EMBO Journal Review Process File - EMBO-2009-71022 Major comment: 1. There is no functional experimentation to show that the dimer interface observed is relevant or correctly described for full-length receptors. Whereas one might be tempted to argue that the dimer interface as defined by crystallography is almost certainly correct, one need only revisit 2003 when this same group reported the dimer interface between two NR1 glycine binding domains. Some months later, the same group reported that the NMDA ligand binding domain dimer was made up of NR1/NR2 (not NR1/NR1). Whereas it may turn out that for some NMDA receptors NR1/NR1 ligand binding dimers are relevant, a bit of caution still might be warranted here. Reply: I am not really sure how to respond to this comment, other than to directly quote from the first paper I believe the referee is referring to (Inanobe et al. Neuron 47 71-84 (2005)) in which we state: 'While of course we have not shown that the NR1 dimer that we see in our crystals of the cycloleucine complex is present in the intact receptor, the fact that we do see such a dimer strongly suggests that in the intact receptors the ligand binding cores are organized as either NR1-NR1 homodimers or NR1-NR2 heterodimers. We would argue that while some experiments suggest the presence of NR1 homodimers (Schorge and Colquhoun, 2003), further experiments are required to unambiguously distinguish between homodimer and heterodimer arrangements.' In our subsequent paper (Furukawa et al. Nature 438 185-192 (2005)) we carry out extensive experimentation to test our hypotheses related to subunit arrangement. Thus I don't think that the referee's comment is either fair or accurate. 2. Overall, the manuscript discusses exciting data describing the crystal structure of the GluR2ATD. The structure is described in detail and structural features of the GluR2-ATD are compared to expected structures of the ATDs of kainate and NMDA receptors based on sequence alignments. However, when the GluR2-ATD structure is used to speculate on the function of the NMDA receptor ATDs and their ligand binding sites, the comparison and the extrapolation of the findings become tenuous. Based on homology models of the ATDs and extensive functional data, several papers from multiple investigators have (already) suggested that Zn2+ and ifenprodil bind in a crevice formed between two lobes in the ATD. The authors did not build homology models of the NR2A-ATD and the NR2B-ATD before mapping the residues important for ligand binding in Figure 8 and consequently, Figure 8 and the discussion concerning this figure do not add important new information to characteristics of the Zn2+ and ifenprodil binding sites that isn't already in the literature. The authors should stick to the data that is included in the paper, which are the crystal structures and the sedimentation equilibrium studies; removal of figure 8 and table 3 would only strengthen the paper by focusing the text on the data at hand. Reply: In accord with this criticism, we have removed Figure 8, Table 3 and the paragraphs that are related to speculations on the binding sites of NMDA receptor allosteric modulators. 3. Following on this theme, the notion that Zn2+ and ifenprodil share a common mechanism of modulation that involves closure of the ATD has been proposed before (see for example Gielen etal., Neuron, 2008). Little is added here to what has been shown in that study. Reply: Consistent with the fact that we have removed almost all of the discussion of NMDA receptors, and taking into account this specific comment, we have modified the sentence related to NMDA modulation (page 13) to read: "In harmony with previously proposed mechanisms of NMDA receptor modulation {Gielen, et al., 2008}, we suggest that the 'weak' L2-L2 interface in the NMDA receptor is akin to that of mGluRs, thereby allowing for movement upon closure of the L1L2 clamshell and allosteric modulation of the ion channel by way of the intervening ligand-binding domains (Figure 8)." © European Molecular Biology Organization 4 The EMBO Journal Review Process File - EMBO-2009-71022 4. The discussion concerning the mechanism by which spermine acts to potentiate NMDA receptors also seems premature. Wouldn't it be better left to future studies to explain this mechanism - perhaps using the GluR2-ATD structure to guide functional experimentation? Reply: In accord with this comment, we have removed the discussion of spermine modulation of NMDA receptors. 5. The authors should also reduce the section on the location of the modulatory bindings sites in NMDA receptors and make sure to cite the appropriate papers that have studied the structural features of Zn2+ and ifenprodil inhibition when they are mentioned. The authors should also cite the papers that have shown that AMPA receptor ATDs are not important for function (for example Pasternack et al., JBC, 2002). Reply: We have removed the section related to NMDA receptor modulator binding sites and have also cited the Pasternack work (on p. 4). 6. The conclusion should be revised. The present study does not "demonstrate the roles that residues, located at the dimer interface, play in defining receptor subtype specific assembly". Rather, the study elaborates on one mechanism by which receptor subtype specific assembly is achieved for the GluR2 AMPA receptor. Demonstrating this would require mutagenesis and experimental data on the assembly of full-length receptors. Furthermore, the sentences on tetramer formation seem to be speculation rather than conclusion. The conclusion should be revised, and the last sentence of the abstract also revised. Reply: We have revised the conclusion to state that our studies simply 'suggest' roles that particular residues may play in subtype specific assembly. We have also changed the portion having to do with tetramer formation to include the notion that other interfaces may be involved in stabilizing the tetramer. The last sentence of the abstract was removed. Minor comments: 6. Page 5, line 11: "GluRD" should be "GluR4" in order to be use a consistent nomenclature throughout the manuscript. Reply: Done. 7. Page 6, give the residues comprising the construct in the main text. Reply: Done (see p. 5) 8. Page 9, line 17: "strand-d" should be "beta4" in order to be use a consistent nomenclature throughout the manuscript. Reply: We believe that we should stick to the "strand-d" wording as it is what the authors of the cited paper use to describe that specific element of structure. Therefore, we have not made this change. 9. Page 14, line 8: "R1 domain" should be "L1 domain". Reply: Done. © European Molecular Biology Organization 5 The EMBO Journal Review Process File - EMBO-2009-71022 Referee #3 (Remarks to the Author): This is a report of the long-awaited structure of the GluR2 ATD. It provides a new breakthrough in the field and will be of considerable interest to the general readership of the EMBO Journal. I have only a few minor suggestions: 1) On page 9, the paragraph comparing the ATD structure with the sequences of AMPA, kainate and NMDA receptors seems out of place. Reply: We agree and have now moved the paragraph to the end of the section. 2) On page 10, I think it is an overinterpretation of the crystal data to suggest that unliganded protein cannot adopt multiple states. All that can be said is that under the conditions that the protein crystallized, only one conformation is seen. Reply: We have modified the sentence to read: "The equivalence in the conformations between the 3 independent dimers thus suggests that under the conditions of crystallization neither the dimer nor the individual subunits readily adopt multiple, unliganded conformational states." 3) On page 19, the authors conclude that tetramerization must derive from the transmembrane domains. I think this also goes beyond what can be reasonably concluded from the data. One can easily envision a model in which very low affinity interactions between ATD dimers, LBD dimers and the transmembrane domains can sum (that is, the sum of the free energies), making a very strong overall interaction. So, whereas their statement may be true, this conclusion cannot be directly made from the data presented in the paper. Reply: We have revised the sentence to read "Tetramer formation in iGluRs, therefore, likely derives from combined interactions between the transmembrane domains together with interactions between the ATD and S1S2 domains." © European Molecular Biology Organization 6