Dear Guillermo, I think the referee has a point because you are comparing two different proteins to propose a model of 2 states even though the enzymes are highly similar with conservation of key features. With 40% id/60% similarity, even if core domain structure and length are conserved, there are likely differences in loops, etc.? Are any such structural variations in the vicinity of the functional site or in regions that could impact approach or function of the active site? If so, the apo and bound forms could also be due to intrinsic differences between the two orthologs?
In the absence of additional supporting evidence, I suppose one way to frame it would be to emphasize that you are proposing a model based on available experimental evidence on closely related proteins and also admit the limitations and unconfirmed/speculative nature of the proposed model? However, I think there may be something more interesting you can do for supporting your theory in case there are other experimental structures of closely related members of the same enzyme family. If so, you could compare in detail additional apo forms of multiple related enzymes and then point to the complex form as an outlier due to its structural differences, which then may represent your proposed model. In addition, do you have assay/mutational analysis of your protein compared to the other one? If so, and if they have similar activity and full conservation of active sites, then that can also be used to support your theory that the 2 enzymes are structurally and functionally very similar and so the two structures are representative of your proposed pathway/model. I had a similar situation a few years ago in comparative structural analysis of 2 nucleases. Active site residues were identical with the same metal and overall architecture was similar despite other structure/dimer differences. Referee insisted we mutate our active site residues and compare, which we did. Hope this helps. Best, Debanu -- Debanu Das, Accelero Biostructures On Tue, Jan 31, 2017 at 9:40 PM, Guillermo Montoya <guillermo.mont...@cpr.ku.dk> wrote: > > Dear all, > > first of all sorry for this off-topic question. I am requesting your help to > find some papers to convince one referee about the comparison > of two different protein states. > > In our manuscript we show the crystal structure of an > enzyme. This structure represents the enzyme after catalysis in complex with > the product. > In the discussion we have superimposed the enzyme in the apo conformation > and the enzyme after catalysis in complex with the product > and we have commented the conformational changes observed > between these 2 states to propose a model. > > The point of the referee is that this comparison is not valid because the > enzymes that we used in the comparison belong to different species. > They are not the same protein. > > However, and this is stated by us in the figs and the manuscript, these two > proteins are > 40% identical and 60% conserved, the polypeptide length is the same, and > the key amino acids and the domain structure are fully conserved. They are > obviously orthologs. > > I´d really appreciate if you can send me some literature/information > to support our approach > > Thanks a lot for your input > > > best > > > > > Guillermo Montoya, Prof., Dr. > Research Director, Protein Structure and Function Programme > Novo Nordisk Foundation Center for Protein Research > Faculty of Health and Medical Sciences, University of Copenhagen, > Blegdamsvej 3A, DK-2200 Copenhagen, Denmark > web: www.cpr.ku.dk > > > >
