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The EMBO Journal Peer Review Process File - EMBO-2011-79645

Manuscript EMBO-2011-79645

A Novel Evolutionarily Conserved Domain of Cell-Adhesion GPCRs Mediates Autoproteolysis Demet Araç, Antony A. Boucard, Marc F. Bolliger, Jenna Nguyen Michael Soltis, Thomas C. Südhof, and Axel T. Brunger

Corresponding author: Axel T. Brunger, Howard Hughes Medical Institute

Review timeline:

Submission date: Editorial Decision: Revision received: Accepted:

26 September 2011 07 November 2011 13 January 2012 16 January 2012

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

07 November 2011

Thank you very much for submitting your research manuscript on crystal structures of the GPCR proteolytic site (GPS) region from adhesion-GPCRs for consideration to The EMBO Journal editorial office. The paper received overall rather positive comments from three expert scientists. Their constructive comments suggest a few experimental additions (points 2 and 3 of ref#1) and otherwise mostly presentational improvements (related to simplifications and better placement of your findings in the more general biological context). I am thus very much looking forward to receive an appropriately revised manuscript that should enable efficient proceedings. Please be still reminded that it is EMBO_J policy to allow a single round of revisions only and that the ultimate decision depends on the content and strength of your adequately modified manuscript. I am very much looking forward to read your revisions. Yours sincerely, Editor The EMBO Journal

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REFEREE REPORTS

Referee #1: This manuscript reports the first crystal structures of GPCR proteolytic site (GPS) motif regions from adhesion GPCRs. The authors find that regions flanking the traditional GPS motif are required for autoproteolysis, and therefore propose the term "GAIN domain" (for "GPCR autoproteolysis inducing domain") to describe the larger domain that includes the traditional GPS motif. Interestingly, the authors provide structural insight into how it is possible to create mutant GAIN domains that do not undergo autoproteolysis but still fold normally, whereas previously it has been widely assumed that mutations interfering with autoproteolysis would also necessarily disrupt proper folding & trafficking. These studies are novel and represent a significant contribution to the field. Specific comments are as follows: 1. The sizes of some of the "cleaved" bands in the Western blots shown in Figure 2 don't seem to make sense. For example, in Fig. 2B, the "cleaved" band is described in the figure legend as being 70 kDa, but on the blot this band appears to be approximately 50 kDa (just above the 47 kDa marker). Is the size mentioned in the text a typo, or are the markers incorrectly indicated on the figure itself? 2. Panel 2D: the CL1-GAIN-Ig fusion protein is described in the text as encompassing residues 533849 of rat CL1, with an Ig tag on the C-terminus and HA tag on the N-terminus. The predicted site of autoproteolysis on this fusion protein is only about 10 amino acids from the C-terminus. Thus, assuming that the authors are visualizing the Ig tag in Fig. 2D (a point that should be clarified in the text), this means that the "cleaved" band should include just the Ig tag plus a few extra amino acids, which should be about 50 kDa. However, the "cleaved" band on the blot looks more like 60 kDa. What accounts for this discrepancy? Also: how do the authors know that the "cleaved" band is truly the result of autoproteolysis? Isn't it possible that this isolated GAIN domain is actually insufficient for autoproteolysis, and that the observed cleavage is the result of proteolysis by other proteases? The authors could more confidently say that this fusion protein is sufficient for autoproteolysis at the GPS motif if they created a mutant fusion protein in which one of the key catalytic residues in the GPS motif (determined from their studies on the full-length receptors) was mutated, with this mutation completely abrogating the formation of the "cleaved" product. 3. Panel 2F: the GPR56 GAIN domain fusion protein is described in the text as encompassing amino acids 115 to 395 of human GPR56, with N-terminal HA and C-terminal Myc tags. As with the CL1 GAIN domain fusion protein, the predicted site of autoproteolysis on the GPR56 fusion protein is only about 10 amino acids from the C-terminus. Thus, since the authors are visualizing the Myc tag in panel 2F, it seems that the "cleaved" band in this instance should include just the Myc tag plus about 10 additional amino acids, which should place the "cleaved" band at less than 5 kDa. However, the "cleaved" band on the blot shown in panel 2F is nearly 20 kDa. What accounts for this discrepancy? Along the same lines as the concerns expressed above (point #2) about panel 2D, it seems that the authors should provide data from additional control experiments to show that the "cleaved" band is absent if they express a mutant GAIN domain that lacks a key catalytic residue in the GPS motif. Otherwise, it seems quite plausible that the "cleaved" bands shown in panels 2D and 2F might not represent evidence of autoproteolysis but instead might simply represent breakdown products caused by other proteases acting on the GAIN domain fusion proteins. 4. Why does the isolated BAI3 GAIN domain not undergo autoproteolysis? To someone who is skeptical about the authors' claim that the GAIN domain is sufficient for autoproteolysis, the observations about the BAI3 GAIN domain might be taken as evidence that GAIN domains in fact are NOT sufficient for autoproteolysis. The authors' model would be more convincing if the authors could mutate one or more residues in the BAI3 GAIN domain (perhaps to make it more like the CL1 GAIN domain) and demonstrate the BAI3 GAIN domain is in fact capable of autoproteolysis with just minimal changes. There also should be some additional discussion as to why cleavage of the BAI3 GAIN domain appears to be dependent on cellular context. Why should the BAI3 GAIN domain undergo cleavage in brain cells but not in HEK-293 cells? The authors do not offer any satisfactory potential explanations for this unusual and striking observation.

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5. In Fig. 4A, it is not clear why the authors show 6 different blots of BAI1, 2 & 3 transfected into HEK-293 cells. Similarly, it is not clear why the authors show 4 different blots of brain tissue in panel 4C. Figure 4 in general is quite confusing and could be greatly simplified by just showing one blot each for BAIs in transfected cells versus brain, unless there is a highly compelling reason for why it is of interest to use numerous different antibodies to show similar patterns of labeling. 6. In describing the data from Figures 8 & 9, the authors state in the text that "Adhesion GPCRs may be hormone receptors", and the legend in Figure 8 states "The HormR domains of cell-adhesion GPCRs may bind hormones to activate the receptor". These statements are quite bold and speculative, relative to the data that are actually shown. The authors should consider softening the language in these locations in the text to something more like: "Adhesion GPCRs exhibit structural similarity to hormone receptors", since no actual binding of hormones to adhesion GPCRs is shown in this manuscript.

Referee #2: The manuscript by Arac and colleagues on the autoproteolysis domain of the cell adhesion GPCRs is a well written manuscript that shows, for the first time, the structure of the GPS entity and the location of this amino acid sequence motif in the context of the three dimensional of the entire GAIN domain. The structures were determined at high resolution and are of excellent stereochemical quality. This work provides new and important insights into the structure of these domains and the relationships between the structure and the mechanism of autoproteolysis as well as the location of residues implicated in various diseases. Comments 1. Based on the data the authors present, how can they conclusively determine that the apparent autoproteolysis is not instead the result, in full or in part, of proteolysis (by another protease) during expression and/or cell lysis? 2. The gel in Fig. 6B is not of publication quality. 3. Fig. 9 is unnecessary, not only because the authors have not presented data that authoritatively defines the location of the GAIN domain relative the GPCR domain, but also because they can simply state the essence of the figure in a simple sentence, i.e. "The GAIN domain is likely located 'above' the GPCR domain..."

Referee #3: Arac et al. report two crystal structures of an evolutionarily conserved domain found not only in cell-adhesion GPCRs but also in polycystic kidney disease proteins. This domain contains what was known as the GPCR proteolysis site (GPS). However, based on the results presented, it is clear that this ~40 residue motif is part of a much larger structural domain, which in its entirety is required for efficient autoproteolysis. The fold of this newly named GAIN domain appears to be novel, and homologues are found in primitive eukaryotes such as slime mold, suggesting an important evolutionarily conserved function for this domain and the autoproteolysis that it catalyzes. The authors map out and analyze the effects of known PKD and cancer associated mutations, and show that many of these interfere with autoproteolysis function, but cancer-associated mutations are more distributed on the surface, suggesting that they may impact other functions of the domain. The GAIN domain was crystallized from both the CL1 and BAI3 GPCRs along with an N-terminal HormR domain. This domain is homologous to a previously determined domain from the corticotrophin release factor receptor (CRFR), but the putative hormone binding site is blocked via its association with the GAIN domain. It is possible that hormones could induce dissociation of these domains, and that GAIN-containing GPCRs represent true hormone responsive GPCRs.

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In summary, this is an excellent paper of high technical merit and thoroughness, and in my opinion is more than appropriate for publication in EMBO J. The methods write up was particularly nice (although perhaps too long for EMBO format). That is not to say that there are not some frustrating aspects to the paper. The CL1 GAIN domain was crystallized in a cleaved state, whereas that of BAI3 was not. The BAI3 also appears to lack an important, presumably catalytic histidine residue next to the proteolysis site, yet in cells BAI3 arrives at the cell surface cleaved. This discrepancy remains unexplained, but it does fortuitously give the authors snapshots of the GAIN domain before and after proteolysis. The even more frustrating aspect (as I am sure it is for the authors too) is that although much was learned about this newly characterized domain and the structural determinants for proteolysis, the role of proteolysis in the function of these proteins remains unknown. It could simply be that it is a strange way that the domain matures into its most stable, functional state. Or it could be much more exciting than that. Because transmembrane signaling by these GPCRs is not understood, it is not possible to know if inhibition of autoproteolysis has any role in transmembrane signaling. The rest of my comments are simply editorial in nature: 1) There are several claims of novelty and "instructions to the establishment" that are out of place and should be eliminated. I'll list some here. Title: "A Novel Evolutionarily Conserved Domain of Cell-Adhesion GPCRs Mediates Autoproteolysis". It is not really a novel domain, is it? Newly characterized, perhaps. After all, the domain has been around for hundreds of millions of years. It seems a title like "Structural characterization of an Evolutionarily Conserved Domain of Cell-Adhesion GPCRs that Mediates Autoproteolysis " would be more accurate and informative. Page 6. " and that our CL1 and BAI3 crystal structures represent the first high-resolution structures of this domain". The authors do not know this for sure. Claiming relevance based on being the first is never good practice in science. Delete "fundamentally" from prior sentence. Page 5. Delete "novel" from "describe a novel, evolutionarily conserved...." Page 15. "This structure was the first structure that was determined at the Stanford Synchrotron Radiation Lightsource by sulfur-SAD phasing using an optimized data collection strategy for a longwavelength at 2 ≈, and one of a few de novo structures that has been determined by sulfur SAD at this resolution." The corresponding author would clearly know if it was the first or not, but I would still omit as it is also irrelevant to the data in the paper. This seems like a statement better saved for a subsequent methodological review. Figure 1 caption. "Note that GPS is defined as a separate domain in the Pfam database (magenta). This definition will need to be revised to include the entire GAIN domain since our crystal structures (see Figure 1B,C) indicate that the GPS motif is not an autonomously folded domain, but rather an integral part of the GAIN domain." Omit second sentence. I don't think this is the place to be giving instructions to the curators of the Pfam database. Page 6. "Thus, the GPS motif itself does not constitute an autonomously folded unit, but rather forms a single folded domain together with the so-called 'stalk' (requiring re-definition in the Pfam database where GPS is defined as a "domain")" Delete the clause in parentheses. 2) Out of place text: Page 9. The comment "In addition to shedding light on the mechanism of autoproteolysis, these uncleavable but folded mutants can be used in future experiments as molecular tools to dissect the functions of CL1 that are autoproteolysis independent." in the results seems out of place, obvious, and can be omitted. I think it is better to stick to the data and not future experiments. Same for the comment on page 10: "This approach may be used for creating uncleavable but folded mutants of other cell-adhesion GPCRs, as well." Or at least move to the discussion.

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3) Figure 1D&E. Please replace these maps with fo-fc omit maps and report contour cutoff. 4) Figure 9. This is a highly speculative figure and there are unexplained features....like the question mark on the right, what are the arrows meant to indicate? Interactions, I suppose, but they are in different places in each view. Is there any evidence in these adhesion receptors that hormones have to bind where indicated? And so on. It is sort of distracting from the main accomplishments of the work, and I would omit. 5) Figure 6. It may be my bias, but it seems the arrows for the labels in panels A and C should be pointing the other way. Aesthetically, the arrows probably shouldn't have different length arrowheads, and the text appears chopped off for some of the labels (A2752... and R276....). I guess I would prefer a better way of indicating where the proteolysis site is than a huge asterisk. It would be better to color the cleaved C-terminal strand uniquely as in previous figures. 6) Figure 8B. Nit-picky, I know, but I think the hormone arrow should be pointing the opposite way. 7) Figure S2C. I am not sure what I am supposed to be getting out of looking at the cleavage site of a completely unrelated autoproteolytic domain. 8) Figure S6A. Arrows. 9) Table S1. Please state resolution range of highest shell of data. The R-factors in the highest shell appear to be essentially random for all the data (>65%). Does this mean the data was anisotropic? If not, then the real resolution limits are probably lower than stated. 10) Table S2&3. These are not tables. There seems to be an orphan reference at the bottom of S3. Maybe it needs to have a "Supplementary References" header?

1st Revision - Authors' Response

13 January 2012

Referee #1: This manuscript reports the first crystal structures of GPCR proteolytic site (GPS) motif regions from adhesion GPCRs. The authors find that regions flanking the traditional GPS motif are required for autoproteolysis, and therefore propose the term "GAIN domain" (for "GPCR autoproteolysis inducing domain") to describe the larger domain that includes the traditional GPS motif. Interestingly, the authors provide structural insight into how it is possible to create mutant GAIN domains that do not undergo autoproteolysis but still fold normally, whereas previously it has been widely assumed that mutations interfering with autoproteolysis would also necessarily disrupt proper folding & trafficking. These studies are novel and represent a significant contribution to the field. Specific comments are as follows:  We thank the reviewer for the positive comments about our work. 1. The sizes of some of the "cleaved" bands in the Western blots shown in Figure 2 don't seem to make sense. For example, in Fig. 2B, the "cleaved" band is described in the figure legend as being 70 kDa, but on the blot this band appears to be approximately 50 kDa (just above the 47 kDa marker). Is the size mentioned in the text a typo, or are the markers incorrectly indicated on the figure itself?  The 47 kDa marker band was misplaced in Figure 2B. This has been corrected in the figure. 2. Panel 2D: the CL1-GAIN-Ig fusion protein is described in the text as encompassing residues © EMBO

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533-849 of rat CL1, with an Ig tag on the C-terminus and HA tag on the N-terminus. The predicted site of autoproteolysis on this fusion protein is only about 10 amino acids from the C-terminus. Thus, assuming that the authors are visualizing the Ig tag in Fig. 2D (a point that should be clarified in the text), this means that the "cleaved" band should include just the Ig tag plus a few extra amino acids, which should be about 50 kDa. However, the "cleaved" band on the blot looks more like 60 kDa. What accounts for this discrepancy? Also: how do the authors know that the "cleaved" band is truly the result of autoproteolysis? Isn't it possible that this isolated GAIN domain is actually insufficient for autoproteolysis, and that the observed cleavage is the result of proteolysis by other proteases? The authors could more confidently say that this fusion protein is sufficient for autoproteolysis at the GPS motif if they created a mutant fusion protein in which one of the key catalytic residues in the GPS motif (determined from their studies on the full-length receptors) was mutated, with this mutation completely abrogating the formation of the "cleaved" product.  In order to confidently say that the CL1 GAIN domain is sufficient for autoproteolysis and that the cleavage product is a result of specific autoproteolysis at the GPS motif, we followed the reviewer’s suggestion and designed two autoproteolysis mutants (H836S and T838G) that were previously demonstrated not to interfere with cell-surface localization and protein folding (Figure 5D). The data shown in the revised Figure 2D and the new Supplementary Figure 2B show that no cleavage occurs for these mutants. Instead, the band that corresponds to the full-length protein becomes stronger, demonstrating that the CL1 GAIN domain is sufficient for autoproteolysis. The calculated molecular weight of the CL1 GAIN domain-Ig fusion protein is approximately 61 kDa (CL1-GAIN=35 kDa, monomeric Ig=26 kDa), but it runs as a 70 kDa protein, presumably due to N-glycosylation at one or more of the five predicted sites in the GAIN domain. Autoproteolysis at the GPS site produces an N-terminal fragment (including an HA epitope) that has a calculated molecular weight of approximately 34 kDa and a C-terminal fragment (containing the last 12 residues of the GAIN domain and the Ig epitope) that has a calculated molecular weight of approximately 27 kDa. The N-terminal cleavage product runs as a 43 kDa protein, again presumably due to glycosylation. As an additional support for the sufficiency of the CL1 GAIN domain for autoproteolysis, our crystal structure of the fragment that contains only the GAIN and HormR domains of CL1 shows that this fragment is autoproteolysed between L837 and T838 (Figure 1D). Moreover, the HormR deletion mutant of the full-length CL1 shows that HormR domain is not required for autoproteolysis (Figure 2B). Taken together, these data show that the CL1 GAIN domain is sufficient for autoproteolysis. 3. Panel 2F: the GPR56 GAIN domain fusion protein is described in the text as encompassing amino acids 115 to 395 of human GPR56, with N-terminal HA and C-terminal Myc tags. As with the CL1 GAIN domain fusion protein, the predicted site of autoproteolysis on the GPR56 fusion protein is only about 10 amino acids from the C-terminus. Thus, since the authors are visualizing the Myc tag in panel 2F, it seems that the "cleaved" band in this instance should include just the Myc tag plus about 10 additional amino acids, which should place the "cleaved" band at less than 5 kDa. However, the "cleaved" band on the blot shown in panel 2F is nearly 20 kDa. What accounts for this discrepancy? Along the same lines as the concerns expressed above (point #2) about panel 2D, it seems that the authors should provide data from additional control experiments to show that the "cleaved" band is absent if they express a mutant GAIN domain that lacks a key catalytic residue in the GPS motif. Otherwise, it seems quite plausible that the "cleaved" bands shown in panels 2D and 2F might not represent evidence of autoproteolysis but instead might simply represent breakdown products caused by other proteases acting on the GAIN domain fusion proteins.  The GAIN domain of mouse GPR56 (residues 115-395) was cloned into the pDisplay vector. The vector encodes an HA tag at the N-terminus, a myc tag at the C-terminus, and the platelet© EMBO

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derived growth factor receptor transmembrane domain (PDGFR-TM) at the C-terminus after the myc tag. The PDGFR-TM is a single transmembrane helix that anchors the fusion protein to the plasma membrane to display it on the cell surface and has a molecular weight of 5.5 kDa. Thus, the C-terminal cleavage product after autoproteolysis in the GPS motif contains last 12 residues of the GAIN domain, the myc tag (10 residues) and the PDGFR-TM (50 residues), adding up to a calculated molecular weight of 8.5 kDa. However, this C-terminal fragment runs as an approximately 15 kDa fragment on the gel. Considering that small proteins often migrate at unpredicted sizes, we believe that this difference is within the expected error. In order to confirm that the cleavage product is a result of the autoproteolysis at the GPS motif and that the GPR56 GAIN domain is sufficient for autoproteolysis, we followed the reviewer’s suggestion and designed two autoproteolysis mutants of the GPR56 GAIN domain (H381S and T383G, homologous residues to the CL1 mutants tested in the revised Fig. 2D). As expected, no cleavage occurs for the mutant proteins (revised Figure 2F). Instead, the band corresponding to the full-length GPR56 GAIN domain became stronger, demonstrating that the GPR56 GAIN domain is sufficient for autoproteolysis. Additionally, we performed surface localization experiments and showed that mutations have no effect on protein folding or localization to the cell-surface (new Supplementary Figure 2C). 4. Why does the isolated BAI3 GAIN domain not undergo autoproteolysis? To someone who is skeptical about the authors' claim that the GAIN domain is sufficient for autoproteolysis, the observations about the BAI3 GAIN domain might be taken as evidence that GAIN domains in fact are NOT sufficient for autoproteolysis. The authors' model would be more convincing if the authors could mutate one or more residues in the BAI3 GAIN domain (perhaps to make it more like the CL1 GAIN domain) and demonstrate the BAI3 GAIN domain is in fact capable of autoproteolysis with just minimal changes. There also should be some additional discussion as to why cleavage of the BAI3 GAIN domain appears to be dependent on cellular context. Why should the BAI3 GAIN domain undergo cleavage in brain cells but not in HEK-293 cells? The authors do not offer any satisfactory potential explanations for this unusual and striking observation.  As demonstrated by our new set of experiments, at least the CL1 and GPR56 GAIN domains are sufficient for autoproteolysis. However, BAIs, even as full-length proteins, are not cleaved significantly in HEK cells. We believe that the lower autoproteolysis efficiency of BAIs compared to CLs is due to intrinsic differences between the proteins. We did not succeed to increase the efficiency of autoproteolysis of BAI by single site mutations. We thus believe that a combination of numerous residues and structural features rather than a few residues (as explained in our manuscript) renders the active site of the CL1 GAIN domain efficient; and that mutating a few residues in BAI3 is not sufficient to make it more autoproteolytic. The concern about why BAIs are cleaved in the brain but not in HEK293 cells is addressed by adding the following sentences to the text: “The conditions of BAI expression (i.e., expression levels, glycosylation machinery, and reductive environment) are very different between insect cells (where we expressed the crystallized proteins) and HEK293 cells on the one hand, and neurons on the other hand. Thus, these differences in post-translational processing and environment could be the reason why BAI3 is uncleaved when expressed in insect or HEK293 cells, but is normally cleaved in brain.“

5. In Fig. 4A, it is not clear why the authors show 6 different blots of BAI1, 2 & 3 transfected into HEK-293 cells. Similarly, it is not clear why the authors show 4 different blots of brain tissue in panel 4C. Figure 4 in general is quite confusing and could be greatly simplified by just showing one blot each for BAIs in transfected cells versus brain, unless there is a highly compelling reason for why it is of interest to use numerous different antibodies to show similar patterns of labeling.

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 There are several reasons why we show multiple blots in figure 4: a) None of our BAI antibodies has been published previously. So, this figure establishes the entire set of antibodies, and demonstrate that they are specific. b) These antibodies were raised against different BAI isoforms, namely BAI1 and BAI3. c) The figure includes important control blots. The GFP blot in panel A is a positive control for transfection and expression of BAIs in HEK cells. The A323 blot in panel C is a negative control for the IP (i.e. the IP was done with a BAI1-specific antibody, 11509, and the precipitated BAI1 could not be detected with the BAI3-specific antibody, A323). Moreover, another result is shown in the A324 blot in panel C; this antibody reacts with an unspecific band in the brain extract (the band just below the FL** band), but antibody 11509 does not precipitate this unspecific band. 6. In describing the data from Figures 8 & 9, the authors state in the text that "Adhesion GPCRs may be hormone receptors", and the legend in Figure 8 states "The HormR domains of celladhesion GPCRs may bind hormones to activate the receptor". These statements are quite bold and speculative, relative to the data that are actually shown. The authors should consider softening the language in these locations in the text to something more like: "Adhesion GPCRs exhibit structural similarity to hormone receptors", since no actual binding of hormones to adhesion GPCRs is shown in this manuscript.  We softened our speculations.

Referee #2: The manuscript by Arac and colleagues on the autoproteolysis domain of the cell adhesion GPCRs is a well written manuscript that shows, for the first time, the structure of the GPS entity and the location of this amino acid sequence motif in the context of the three dimensional of the entire GAIN domain. The structures were determined at high resolution and are of excellent stereochemical quality. This work provides new and important insights into the structure of these domains and the relationships between the structure and the mechanism of autoproteolysis as well as the location of residues implicated in various diseases.  We thank the reviewer for the positive assessment of our work. Comments 1. Based on the data the authors present, how can they conclusively determine that the apparent autoproteolysis is not instead the result, in full or in part, of proteolysis (by another protease) during expression and/or cell lysis?

 In the literature, several groups have independently reported compelling evidence that various cell-adhesion GPCRs undergo autoproteolysis (see Krasnoperov et al, 1997; Ichtchenko et al, 1999; Chang et al, 2003 and especially Lin et al., 2004, where they focused on this question.). Our structural and functional data agree with the previously proposed autoproteolytic mechanism (Figure 5). In addition, our data suggest that it is unlikely that another protease is involved: a) The structures of both CL1 and BAI3 GAIN domains show that the scissile bond is buried inside the core of the protein and would be inaccessible to another protease.

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b) Among unfolded CL1 mutants, none of them are cleaved. In contrast, all mutants that are cleaved are folded, suggesting that the protein has to be folded for being cleaved. This observation is consistent with the autoproteolysis hypothesis that requires proper positioning of numerous residues at the autoproteolytic site, but is inconsistent with the involvement of another protease. 2. The gel in Fig. 6B is not of publication quality.  Human PKD1 is an approximately 500 kDa protein with numerous glycosylation sites, disulfide bonds and 11 transmembrane helices. The large size of the expression vector (20,000 bp) decreases the transfection efficiency in HEK293 cells. The large size of the protein makes this protein very difficult to work with. We have systematically attempted to obtain better looking gels by changing the DNA concentration, transfection method, western blot membrane type, pore size, blotting time etc. but could not improve the appearance of the gels. However, all controls were performed to ensure the validity of our results and our results were always reproducible (note the disappearance of the strong C-terminal cleavage product). In addition, we show supplementary data consisting of western blots for the N-terminal GFP tag (Supplementary Figure 5A). 3. Fig. 9 is unnecessary, not only because the authors have not presented data that authoritatively defines the location of the GAIN domain relative the GPCR domain, but also because they can simply state the essence of the figure in a simple sentence, i.e. "The GAIN domain is likely located 'above' the GPCR domain..."  We revised Figure 9 to address reviewer 2 and reviewer 3’s concerns and clearly state that it represents only one of the possible orientations of the GAIN domain with respect to the transmembrane helices. We believe that the revised figure (which is drawn to scale) provides a useful guide for the reader to accompany our discussions. Referee #3: Arac et al. report two crystal structures of an evolutionarily conserved domain found not only in cell-adhesion GPCRs but also in polycystic kidney disease proteins. This domain contains what was known as the GPCR proteolysis site (GPS). However, based on the results presented, it is clear that this ~40 residue motif is part of a much larger structural domain, which in its entirety is required for efficient autoproteolysis. The fold of this newly named GAIN domain appears to be novel, and homologues are found in primitive eukaryotes such as slime mold, suggesting an important evolutionarily conserved function for this domain and the autoproteolysis that it catalyzes. The authors map out and analyze the effects of known PKD and cancer associated mutations, and show that many of these interfere with autoproteolysis function, but cancer-associated mutations are more distributed on the surface, suggesting that they may impact other functions of the domain. The GAIN domain was crystallized from both the CL1 and BAI3 GPCRs along with an N-terminal HormR domain. This domain is homologous to a previously determined domain from the corticotrophin release factor receptor (CRFR), but the putative hormone binding site is blocked via its association with the GAIN domain. It is possible that hormones could induce dissociation of these domains, and that GAIN-containing GPCRs represent true hormone responsive GPCRs. In summary, this is an excellent paper of high technical merit and thoroughness, and in my opinion is more than appropriate for publication in EMBO J. The methods write up was particularly nice (although perhaps too long for EMBO format).

 We very much thank the reviewer for the positive assessment of our work .

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That is not to say that there are not some frustrating aspects to the paper. The CL1 GAIN domain was crystallized in a cleaved state, whereas that of BAI3 was not. The BAI3 also appears to lack an important, presumably catalytic histidine residue next to the proteolysis site, yet in cells BAI3 arrives at the cell surface cleaved. This discrepancy remains unexplained, but it does fortuitously give the authors snapshots of the GAIN domain before and after proteolysis.

 We are not sure if there is a misunderstanding or a typo in reviewer’s comment. BAI3 arrives at the HEK cell surface “uncleaved”, not “cleaved”; suggesting that its autoproteolysis is not required for plasma membrane localization. This result (lack of autoproteolysis in HEK cells) is consistent with the lack of autoproteolysis in the crystal structure of the BAI3 protein (that was expressed in insect cells). However, we show that endogenous BAI proteins in brain are largely cleaved. We believe that the autoproteolysis efficiency of BAIs is much less than that of CLs due to intrinsic differences between proteins (for example the presence of an Arg instead of a His residue is one of the many reasons why BAIs are less efficient). “The conditions of BAI expression (i.e., expression levels, glycosylation machinery, and reductive environment) are very different between insect cells and HEK293 cells on the one hand, and neurons on the other hand. Thus, these differences in posttranslational processing and environment could be the reason why BAI3 is uncleaved when expressed in insect or HEK293 cells, but is normally cleaved in brain.” We have revised the text accordingly. The even more frustrating aspect (as I am sure it is for the authors too) is that although much was learned about this newly characterized domain and the structural determinants for proteolysis, the role of proteolysis in the function of these proteins remains unknown. It could simply be that it is a strange way that the domain matures into its most stable, functional state. Or it could be much more exciting than that. Because transmembrane signaling by these GPCRs is not understood, it is not possible to know if inhibition of autoproteolysis has any role in transmembrane signaling.

 We agree that the exact role of proteolysis in the function of these proteins remains unknown (as well as the function of these proteins themselves). Still, the conservation of the GAIN domain throughout evolution and its involvement in multiple diseases suggest an important role. Understanding the signaling pathways of cell-adhesion GPCRs is a long-term goal that may eventually reveal the role of autoproteolysis in the GAIN domain. The rest of my comments are simply editorial in nature: 1) There are several claims of novelty and "instructions to the establishment" that are out of place and should be eliminated. I'll list some here. Title: "A Novel Evolutionarily Conserved Domain of Cell-Adhesion GPCRs Mediates Autoproteolysis". It is not really a novel domain, is it? Newly characterized, perhaps. After all, the domain has been around for hundreds of millions of years. It seems a title like "Structural characterization of an Evolutionarily Conserved Domain of Cell-Adhesion GPCRs that Mediates Autoproteolysis " would be more accurate and informative.  We have replaced "novel" with " newly identified" throughout the manuscript, but would prefer to keep the title as is because we did, after all, discover a previously unidentified domain that exhibits interesting features, and would like to communicate this fact in the title. Page 6. " and that our CL1 and BAI3 crystal structures represent the first high-resolution structures of this domain". The authors do not know this for sure. Claiming relevance based on being the first is never good practice in science. Delete "fundamentally" from prior sentence.

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 “fundamentally new” has been replaced with "newly identified". We state the date of the DALI search that documents the novelty of the new fold. Page 5. Delete "novel" from "describe a novel, evolutionarily conserved...."  “Novel” has been replaced with "newly identified" Page 15. "This structure was the first structure that was determined at the Stanford Synchrotron Radiation Lightsource by sulfur-SAD phasing using an optimized data collection strategy for a longwavelength at 2 Å, and one of a few de novo structures that has been determined by sulfur SAD at this resolution." The corresponding author would clearly know if it was the first or not, but I would still omit as it is also irrelevant to the data in the paper. This seems like a statement better saved for a subsequent methodological review.  We are providing the detailed methodology in the methods section that describes our data collection and structure solution strategy, so there is no need for a methods paper. Figure 1 caption. "Note that GPS is defined as a separate domain in the Pfam database (magenta). This definition will need to be revised to include the entire GAIN domain since our crystal structures (see Figure 1B,C) indicate that the GPS motif is not an autonomously folded domain, but rather an integral part of the GAIN domain." Omit second sentence. I don't think this is the place to be giving instructions to the curators of the Pfam database.  We reworded this sentence to simply state the fact that the GPS motif is not an autonomously folded domain contrary to the current definition in the Pfam database. Page 6. "Thus, the GPS motif itself does not constitute an autonomously folded unit, but rather forms a single folded domain together with the so-called 'stalk' (requiring re-definition in the Pfam database where GPS is defined as a "domain")" Delete the clause in parentheses.  This sentence has been removed from the text. 2) Out of place text: Page 9. The comment "In addition to shedding light on the mechanism of autoproteolysis, these uncleavable but folded mutants can be used in future experiments as molecular tools to dissect the functions of CL1 that are autoproteolysis independent." in the results seems out of place, obvious, and can be omitted. I think it is better to stick to the data and not future experiments. Same for the comment on page 10: "This approach may be used for creating uncleavable but folded mutants of other cell-adhesion GPCRs, as well." Or at least move to the discussion. These sentences have been removed from the text. 3) Figure 1D&E. Please replace these maps with fo-fc omit maps and report contour cutoff.

 We thank the reviewer for the suggestion. We now state the contour level for Figures 1D and E. Instead of replacing these figures with omit maps, we chose to present density-modified

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experimental (SAD) electron density in Supplementary Figure 7 in addition to the existing figures. Since these maps were calculated without reference to a model, they are even more stringent than an omit map. The density-modified maps clearly show that the CL1 structure is cleaved, whereas the BAI3 is uncleaved. 4) Figure 9. This is a highly speculative figure and there are unexplained features....like the question mark on the right, what are the arrows meant to indicate? Interactions, I suppose, but they are in different places in each view. Is there any evidence in these adhesion receptors that hormones have to bind where indicated? And so on. It is sort of distracting from the main accomplishments of the work, and I would omit.

 We revised Figure 9 to address reviewer 2 and reviewer 3’s concerns. In particular, we now state that our model represents only one of the possible orientations of the GAIN domain with respect to the transmembrane helices. 5) Figure 6. It may be my bias, but it seems the arrows for the labels in panels A and C should be pointing the other way. Aesthetically, the arrows probably shouldn't have different length arrowheads, and the text appears chopped off for some of the labels (A2752... and R276....). I guess I would prefer a better way of indicating where the proteolysis site is than a huge asterisk. It would be better to color the cleaved C-terminal strand uniquely as in previous figures.

 These corrections have been made. 6) Figure 8B. Nit-picky, I know, but I think the hormone arrow should be pointing the opposite way.  The arrow direction has been changed. 7) Figure S2C. I am not sure what I am supposed to be getting out of looking at the cleavage site of a completely unrelated autoproteolytic domain.  This panel has been removed from the figure. 8) Figure S6A. Arrows.  The arrow direction has been changed. 9) Table S1. Please state resolution range of highest shell of data. The R-factors in the highest shell appear to be essentially random for all the data (>65%). Does this mean the data was anisotropic? If not, then the real resolution limits are probably lower than stated.  We apologize for the omission - the resolution ranges of the highest shell of data are now stated in the footnote of Supplementary Table 1. We chose to include this highest resolution bin since I/σ(I) is still reasonable in that shell with excellent redundancy. Furthermore, upon refinement, Rfree (Supplemental Table 1) and σA (~ 0.87 for CL1 and 0.83 for BAI3) in the highest resolution bin are acceptable, indicating that the data is more accurate in that bin as might be suggested by the high Rsym (a consequence of the excellent redundancy of the data sets).

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10) Table S2&3. These are not tables. There seems to be an orphan reference at the bottom of S3. Maybe it needs to have a "Supplementary References" header?  We defer to EMBO editorial staff on the question of how to references tables. The reference has been corrected.

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