Reviewer 1: Lakshminarayan Iyer, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, USA
This is a previously uncharacterized, circularly permuted subfamily of the Ras GTPases that is sporadically distributed in a wide phyletic range of eukaryotes. The observation merits publication in order to bring attention to this subfamily of proteins. I have a few points and several editorial suggestions.
- The domain architectures need to be precisely described and defined. There should only be one standard name for domains. Sec23/Sec24 trunk is the vwA domain.
Authors' response: These two names indeed designate the same domain. We have revised the respective part of the text so that this is now clearer. However, because the different names for this same domain are used variously in standard databases (SMART, Pfam), we believe it is appropriate to mention both.
COG names are useful when the domain function or fold is not clear. In the case of COG2373, there is little doubt as to what it is and it should be correctly represented.
The alpha-2 macroglobulin domain needs to be more precisely described and it definitely is not alpha helical. The cpRAS domain is lodged within multiple repeats of the macroglobulin-like domain; this part is not described or analyzed correctly.
Authors' response: It is true that COG2373 proteins and DdiCPRas2 share several domains with the alpha-2 macroglobulin family of serum proteins, so we have added details on this to the respective paragraph and to Fig. 2B. It is possible that COG2373 proteins are bacterial orthologs of the alpha-2 macroglobulin family, but the similarity is low and to our best knowledge, no COG2373 protein has been functionally characterised, so the actual nature of these proteins remains uncertain. In addition, the bacterial COG2373 proteins and DdiCPRas2 exhibit regions of homology extending beyond the alpha-2 macroglobulin domains and this fact is well captured by the concept of the COG2373 family as defined in the standard database of conserved domains and families – the CDD database. We have therefore kept the reference to the COG2373 family in the revised text, though with doubts expressed about the annotation of the family („Large extracellular alpha-helical protein").
Please check the architectures, some versions fused to the vWA have an additional ring finger.
Authors' response: We are very thankful for this point, we indeed missed a RING finger motif at the C-terminus of the CPRas protein from Naegleria gruberi. In addition, further inspection of CPRas protein sequences revealed a possible zinc finger upstream the VWA domain. We have added a note on this into the revised text.
- Given its solo presence, could DdiCPRas2 be an artificial fusion caused by misassembly? Such things are very common with genomic data. Please check and confirm.
Authors' response: A protein with the same domain architecture is encoded by the genome of another Dictyostelium – D. purpureum. This gene was omitted from analyses described in the paper, because the sequence available at the moment is incomplete, but we have made a note on it in the revised manuscript, so the authenticity of DdiCPRas2 is now beyond any doubt.
- Too much is being made of Anand et al's. statement about the existence of a single circularly permuted GTPase family.
Authors' response: We follow the classical Popperian view on how science works. Anand et al. elaborated a hypothesis explaining why they had found only the G4-G5-G1-G2-G3 permutation of the GTPase domain (see also the expanded discussion on their claims incorporated into the revised manuscript upon suggestion of the second reviewer). This hypothesis should be taken seriously until evidence is found which would falsify it. This is exactly what our paper does.
Circularly permuted proteins are observed across a wide range of protein folds and its emergence is solely determined by natural selection. I suggest that, it be mentioned earlier during the description of discovery of the circular permutation.
Authors' response: We do not feel there is enough evidence supporting the notion that the emergence of circularly permuted proteins is solely determined by natural selection. We are rather inclined to the view that most such rearrangements are removed by negative selection and those that survive are mostly selectively neutral. However, it is true that circular permutations occur widely, so we have inserted a note on this into the first paragraph.
- I don't see why pathogen defense needs to be invoked as a specialized process in which the cpRAS is involved and perhaps some elaboration is needed.
Authors' response: The reviewer may be right in that the speculation on the potential involvement of CPRas proteins in pathogen defence is unsubstantiated. We have deleted the respective section from the revised manuscript and leave the possibilities on the function of CPRas proteins completely open.
Other minor and editorial comments for the authors:
- In the description of the swap and the circular permutation, it should be clarified that the definitions are only for the protein domain and not the whole protein (which could have multiple domains)
Authors' response: There is probably some misunderstanding here. The cited papers by the Unger's group [3, 4]define the terms „circular permutations" and „swap" with respect to full proteins and make no reference to protein domains. We regard such definitions impractical, because they obscure the fact that domains are the actual basic units from which proteins are built. Following these definitions, the relationship between canonical GTPases (like HRAS) and CPRas proteins would be called „swap" without the understanding that it is nothing more than „circular permutations" of one particular domain (the GTPases domain). That's why we explicitly explain (see the last sentence of the first paragraph) that we apply the term „circular permutation" to the level of protein domains.
- Last line of page 1:"..individual protein domains, regardless "of" the ..."
Authors' response: The grammatical error has been corrected.
- Shouldn't RAS be written as Ras?
Authors' response: There is little consensus in the literature how the names of these GTPases should be written; all the forms „RAS", „Ras", or „ras" have been used in various sources, often with different meanings. We keep „RAS family" as a name for a broader group of GTPases comprising multiple subgroups as Ras, Rap, Rheb, CPRas etc. The name of the one specific representative of the family, the human HRAS, follows the standard HUGO nomenclature of human genes/proteins.
- Page 3 last paragraph: Instead of opisthokont, use ichthyosporean as the latter is more precise.
Authors' response: We do not think that it is more precise to treat Capsaspora owczarzaki as an ichthyosporean. It's true that it is classified in Ichthyosporea in the NCBI taxonomic scheme, but other authorities place the organism outside Ichthyosporea as a separate opisthokont lineage (e.g., Adl et al., J Euk Microbiol 2005, 52: 399–451). We therefore keep the designation „unicellular opisthokont" in the revised text.
- Page 3. last paragraph: What is "global structure?", perhaps "domain architecture" is meant.
Authors' response: Yes, we meant domain architecture. This expression is now used in the revised text, as it seems to be more appropriate.
- Figure 2B. Revise according to the suggestions above.
Authors' response: The figure has been revised accordingly.
- I don't see the point in Table S1. I think the descriptions are clear enough.
Authors' response: The text and the Figure 1mention only the whole eukaryotic clades for which evidence for the cpRAS domain is missing. We believe that it is useful to specify the actual species lacking the cpRAS domain, so we decided to retain the Table S1.
Reviewer 2: Fyodor Kondrashov, Section on Ecology, Behavior and Evolution, Division of Biological Sciences, University of California at San Diego, USA
This manuscript presents straightforward evidence in support of new RAS-like (cpRAS) domain with a distinct order of conserved GTPase motifs distinct from what has been claimed as the only possible arrangement found in YRG family of proteins. The manuscript also does a good job with exploring the phylogenetic patterns of the cpRAS domain and the possible functional implications. The phyletic pattern, which apparently involves multiple losses and horizontal gene transfers is intriguing, although is not unique. It would be interesting to see whether or not this domain could have evolved multiple times independently, however, this possibility is unlikely given that the circular permutation of the GTPase domains seems very similar between different species.
Authors' response: We actually mention phylogenetic evidence for a single origin of the cpRAS domain in the text.
It was not clear to me why the other authors decided that the YGR arrangement of the GTPase motifs was the only one possible. Perhaps a short discussion would be appropriate for the uninitiated readers.
Authors' response: We have added to the third paragraph two sentences specifying the argument raised by Anand et al. against the existence of permutations other than G4-G5-G1-G2-G3.
With this regard I would change the first sentence of the abstract to something like "A recent systematic survey claimed (or suggested) that the YRG (or YawG/YlqF) family with the G4-G5-G1-G2-G3 order of the conserved GTPase motifs represents the only possible circularly permuted variation of the canonical GTPase structure."
Authors' response: We have modified the first sentence of the abstract following the reviewer's suggestion.