The UBR-box and its relationship to binuclear RING-like treble clef zinc fingers
© Kaur and Subramanian. 2015
Received: 9 March 2015
Accepted: 2 July 2015
Published: 17 July 2015
The N-end rule pathway is a part of the ubiquitin–dependent proteolytic system wherein N-recognin proteins recognize the amino terminal degradation signals (N-degrons) of the substrate. The type 1 N-degron recognizing UBR-box domain of the eukaryotic Arg/N-end rule pathway is known to possess a novel three-zinc-stabilized heart-shaped fold.
Using sequence and structure analysis we argue that the UBR-box fold emerged from a binuclear RING-like treble clef zinc finger. The RING-like core is preserved in the UBR-box and the metal-chelating motifs display significant sequence and structural similarity to B-box and ZZ domains. UBR-box domains retrieved in our analysis co-occur with a variety of other protein domains, suggestive of its involvement in diverse biological roles. The UBR-box is a unique family of RING-like treble clefs as it displays a distinct circular permutation at the zinc-knuckle of the first zinc-binding site unlike other documented permutations of the RING-like domains which occur at the second zinc-binding site. The circular permutation of the RING-like treble clef scaffold has possibly aided the gain of a novel and relatively deep cleft suited for binding N-degrons. The N- and C-terminal extensions to the circularly permuted RING-like region bind a third zinc ion, which likely provides additional stability to the domain by keeping the two halves of the permuted zinc-knuckle together.
Structural modifications and extensions to the RING-like core have resulted in a novel UBR-box fold, which can recognize and target the type 1 N-degron containing proteins for ubiquitin-mediated proteolysis. The UBR-box appears to have emerged during the expansion of ubiquitin system pathway-related functions in eukaryotes, but is also likely to have other non-N-recognin functions as well.
This article was reviewed by Eugene Koonin, Balaji Santhanam, Kira S. Makarova.
KeywordsBinuclear treble clefs zinc fingers B-box ZZ domain N-end rule novel fold U-box
The N-end rule pathway relates the biological half-life of cellular proteins to the presence of N-terminal destabilizing signals (N-degrons), which upon being recognized by N-recognins, targets the substrate protein for proteolysis . In eukaryotes, two branches of the N-end rule pathway, viz., the Arg/N-end rule and the Ac/N-end rule recognize and target different sets of N-degrons . While the Arg/N-end rule pathway recognizes N-terminal arginylated residues and N-terminal basic unmodified residues as N-degrons, the Ac/N-end rule pathway recognizes small uncharged acetylated N-terminal residues . Although, differing in the kind of recognized N-degron and the steps involved in their processing, both branches lead to a common ubiquitin mediated-proteasomal degradation of their targets .
Many binuclear treble clefs such as the B-box, ZZ, Zf-UBP, TFIIH-p44 and C1-type zinc fingers share two characteristic features with the RING finger. First, these domains contain a three-stranded β-sheet comprising of the primary β-hairpin and the C-terminal β-strand . Second, the position of the last metal-chelating residue is invariably at the beginning of the C-terminal β-strand . Based on the presence of these features, the aforementioned binuclear treble clefs are suggested to comprise a monophyletic group , which we refer to as the binuclear RING-like treble clef domains. RING finger domains from a number of proteins have been shown to function as E3 ubiquitin ligases wherein they mediate protein-protein interactions with E2 ubiquitin ligases . Besides the well acknowledged role of RING fingers in the ubiquitination pathway, the B-box and the Zf-UBP are also known to function as E4 ubiquitin ligases  and as ubiquitin-binding modules in the aggrosome pathway , respectively. Our analysis suggests that the UBR-box domain is yet another novel variant of the RING-like treble clef which functions in the ubiquitin system pathway.
Results and discussion
The UBR-box may be regarded as a novel three zinc-stabilized fold [4, 5], but a closer inspection reveals striking resemblances to the B-box (Fig. 2c) and ZZ domains (Fig. 2d). On comparison of the structures of the UBR-box with representatives of the B-box, ZZ domain and RING finger (Fig. 2b-e), we observe that the UBR-box core is related by a circular permutation to the RING-like treble clef domain (Fig. 2e) with additional N- and C-terminal extensions that bind a third zinc ion (Fig. 2f). The UBR-box lacks a well-defined second β-strand in the primary β-hairpin of the treble clef and instead harbors a loop at this position. This loop hydrogen bonds with the first β-strand near the first-turn of the α-helix, thus, forming a short region characteristic of the primary β-hairpin of treble clefs (PDB identifiers 3NIT_A, 3NY1_B). Apart from these minor structural changes, the UBR-box has all characteristic features of the RING-like treble clefs .
The sequences of the UBR-box classified in Pfam (PF02207) and those obtained by the JackHMMER search were analyzed for the presence of co-occurring domains and their distribution across eukaryotes (see Additional file 1 for details). We observe a widespread distribution of the UBR-box and their co-occurrence with a variety of other domains (Additional file 2: Figure S1). We could detect the UBR-box domain in Amoebozoa, Euglenozoa, Diplomonadida, Choanoflagellida, Parabasalia, Heterolobosea, Stramenopiles, Alveolata, Haptophyceae, Ichthyosporea, Fungi, Viridaeplantae and Metazoa. Our analysis suggests that in a majority of proteins, the UBR-box is the only domain identified by automated searches. The commonly co-occurring domains with the UBR-box include the RING, HECT, ZZ, PHD, ClpS and F-box domains. In some of the retrieved sequences, domains such as protein kinase (PF00069), methyltransferase (PF10294, PF05050), zf-RanBP (PF00641), telomerase RNA binding domain (PF12009), DNA polymerase processivity factor (PF02916), pectate lyase (PF12708), concanavalin A-like lectin/glucanases (PF13385), siroheme biosynthesis protein (PF14824, PF14823), oxysterol-binding protein (PF01237), etc., are also present adjacent to the UBR-box domain (Additional file 2: Figure S1).
Previous reports have shown that UBR-box containing proteins function in diverse biological processes [2, 19, 20] such as degradation of misfolded proteins , chromosome segregation , meiosis [23, 24], spermiogenesis , neurogenesis , apoptosis , cardiovascular development , sensing of heme , oxygen , nitric oxide , and short peptides , quorum sensing , the regulation of peptide import [33, 34], regulation of pancreatic and brain function and development , and senescence, germination and hypoxia in plants [35, 36]. The UBR-box domain is referred to as a structural scaffold with varying binding specificities  as not all the UBR-box domains interact with N-degrons [1–3] and may bind other moieties which are not necessarily substrates for ubiquitination. For example, binding of short peptides to the UBR-box domain of S. cerevisiae UBR1 is known to allosterically regulate the activity of adjacent domains [31, 34]. Currently, the biological function and interacting partners for most non-N-recognin UBR-box domains have not been experimentally demonstrated. A recent report reveals that the UBR-box domain in Drosophila non-N-recognin UBR3 can bind and regulate the activity of Drosophila inhibitor of apoptosis protein 1 (DIAP1) . The large repertoire of co-occurring domains (Additional file 2: Figure S1) identified in our analysis is suggestive of diverse biological roles for the UBR-box.
The UBR-box domain is not classified by SCOP  or CATH , but has been grouped under the B-box zinc-binding domain-like X-group of ECOD  based on structural similarity (ECOD identifier e3ny1A1). Dali structure similarity searches  initiated using the human or S. cerevisiae UBR-box against the PDB did not find matches to any other proteins as reported earlier [4, 5]. However, TopSearch , could identify several mononuclear and binuclear treble clefs, including B-box and ZZ domain, as matches to the structure of UBR-box (PDB identifier 3NY1_A). The best match is that of the mononuclear treble clef domain of Poly(ADP-ribose) polymerase 1 (PARP1; PDB identifier 4AV1_A) with a similarity score of 45.1 and a RMSD of 2.3 Å over an alignment length of 50 Cα atoms with a single permutation at the zinc-knuckle. Interestingly, TopSearch  could identify the exact site of circular permutation while superimposing the UBR-box with other treble clef domains. Automated and manual pairwise structural superimposition of the UBR-box with B-box and ZZ domains also reveal structural similarity among these treble clefs and they could be aligned over the full length of their corresponding binuclear RING-like regions upon assuming circular permutation at the zinc-knuckle. For example, using TM-Align , the circularly permuted RING-like region of UBR-box (PDB identifier 3NY1_A) could be superimposed on the B-box domain (PDB identifier 3DDT_A) with an RMSD of 2.7 Å over 38 Cα atoms with a TM-Align score of 0.44 (normalized over the length of the B-box). The circularly permuted binuclear RING-like region of UBR-box (PDB identifier 3NY1_A) and the B-box domain (PDB identifier 3DDT_A) could be manually superimposed using the pair fitting command of PyMOL with an RMSD of 2.46 Å over 40 pairs of Cα atoms (Additional file 3: Figure S2). Thus, the structural and sequence similarities discussed above suggest an evolutionary connection between the UBR-box and binuclear RING-like treble clefs.
The third zinc ion of the UBR-box, chelated by three ligands from the C-terminal extension in human UBR1 and a shared ligand with the first zinc ion, is important for the N-degron-binding function [4, 5] and perhaps provides additional structural stabilization to the circularly permuted zinc-knuckle. Circular permutations to the core of a treble clef fold are rare unlike their relatively common occurrence in the zinc ribbon fold [10, 45], and a previously documented example of circularly permuted treble clef is that of the C-terminal domain of prolyl-tRNA synthetase (PDB identifier 1H4Q_A) [10, 46]. However, we note the presence of permuted treble clefs in other structures such as the triquetra knot containing protein Rds3p (PDB identifier 2K0A_A; ECOD identifiers e2k0aA1, e2k0aA2, e2k0aA3) (Additional file 4: Figure S3). Circular permutations to the RING-like treble clefs have been observed in C1 and TFIIH-p44 zinc finger domains [10, 8] (Additional file 5: Figure S4) but in these structures the permutation occurs at the zinc ribbon-like region, which harbors the second zinc-binding site. Circular permutation at the first zinc-binding site of binuclear RING-like treble clefs is unique to the UBR-box, though a similar permutation is seen in one other mononuclear treble clef, viz., the Rds3p, which has three treble clef motifs of which two are circularly permuted (PDB identifier 2K0A_A) (Additional file 4: Figure S3) . The third zinc ion of the UBR-box, which shares a metal-chelating residue with the first zinc ion of the treble clef, forming a two-metal ion cluster is also not unprecedented. Such two-metal ion clusters are observed in other treble clefs, such as the RING finger of RAG1 (PDB identifier 1RMD_A) and FYVE/PHD zinc finger from the C-terminal region of E3 ubiquitin-protein ligase CHFR (PDB identifier 2XOC_A). Thus, we observe that the structural modifications to the treble clef fold of the UBR-box have precedents and these further corroborate the relationships between the UBR-box and RING-like domains.
Based on sequence and structural similarities of their binuclear regions, we hypothesize that the UBR-box plausibly emerged via circular permutation of a RING-like treble clef scaffold. The circular permutation likely resulted in the splitting of the treble clef’s zinc-knuckle and joining of the C-terminal β-strand to the first-half of the split knuckle. This in turn, resulted in the formation of a relatively deep-binding cleft on which the N-degron-recognizing and -binding functions likely emerged. The extensions to the RING-like core perhaps developed subsequently, to provide additional structural stability to the permuted zinc-knuckle. These extensions are also important for function as they harbor residues that help form an acidic cleft to bind the basic type 1 N-degrons [4, 5]. A comparison of the UBR-box from human and S. cerevisiae UBR1 revealed that one of the ligands for the third zinc ion (His166 in human UBR1, PDB identifier 3NY1_A and His118 in S. cerevisiae UBR1, PDB identifier 3NIH_A) while being present at an equivalent spatial location, is circularly permuted with respect to its position in the sequence (Fig. 1a,b). In the human UBR-box, this His is present on the C-terminal extension of the UBR-box, whereas in the S. cerevisiae protein, it is located at the N-terminal region. It has been noted previously that all zinc-chelating residues with the exception of this His are conserved among human UBR proteins . However, we observe the presence of other metal-chelating aminoacids in the N-terminal extensions of the RING-like core of the UBR-box, which are positioned equivalent to the S. cerevisiae N-terminal His in the sequence and may help chelate the third zinc ion (for example, Cys1174 in human UBR5, UniProt ID: O95071, Cys1658 in human UBR4, UniProt ID: Q5T4S7 in Fig. 3).
It is believed that metal chelation aided the emergence of new protein folds, concomitant with the advent and expansion of novel functions in eukaryotes . Also, it is known that many binuclear treble clefs were already present before the emergence of the Last Eukaryotic Common Ancestor (LECA) and at least one binuclear treble clef was present in the Last Universal Common Ancestor (LUCA) . It has been suggested that some of the binuclear RING-like treble clefs present in the LECA diverged into new superfamilies in eukaryotes and gained distinct biochemical functions [9, 48]. The B-box is one such binuclear RING-like treble clef, which is present in both prokaryotes and eukaryotes where it is involved in the folding, stability and proteolysis-related functions of membrane proteins, and in ubiquitin system pathway-related functions, respectively . The ZZ domain on the other hand is present only in eukaryotes [49, 50] where it is proposed to play a scaffolding role and mediate protein-protein interactions . The B-box and the ZZ domain share sequence and structural similarities over the full length of the binuclear treble clef domain  and are likely related in evolution. The UBR-box appears to have diverged only recently from the binuclear RING-like treble clef group given that it is present only in eukaryotes [49, 2, 1] where it functions in the Arg/N-end rule pathway . A recent emergence of the UBR-box from RING-like treble clefs is also supported by similar patterns of emergence of other components of the Arg/N-end rule pathway . For example, Arg-conjugating enzymes that lead to the addition of primary destabilizing N-terminal Arg on secondary destabilizing N-terminal residues are likewise confined to eukaryotes .
Intriguingly, type 2 N-degron recognizing PRT1 protein from A. thaliana is proposed to interact with the aromatic N-terminal residue of the type 2 N-degron via its ZZ domain . Although not very well characterized, ZZ-domains are suggested to have two surfaces, a hydrophobic patch and an acidic region, which could likely interact with other proteins . Some of the conserved hydrophilic and hydrophobic aminoacids are at a similar structural location as the region utilized by the UBR-box to interact and bind its substrate. A comprehensive survey of the various binding surfaces used by mononuclear  and binuclear treble clefs has been reported earlier . However, the mode of binding the N-degron peptide in a relatively deep cleft within the treble clef domain, where the peptide forms antiparallel β-sheet interaction  with the zinc-knuckle containing β-strand, is observed only in the UBR-box. The backbone atoms of the first three residues and the side chains of the first two residues of the N-degron contact the UBR-box [5, 4]. The N-terminal basic residue is accommodated in an acidic cleft formed by residues contributed primarily by the first-half of the split zinc-knuckle and the C-terminal extension . Additionally, residues on the primary β-hairpin and the first-half of the split zinc-knuckle are involved in hydrogen bonds and hydrophobic interactions with the N-degrons . A similar binding pocket is seen in the PARP1 mononuclear treble clef (PDB identifier 4AV1_A), which recognizes and binds dsDNA  (Additional file 6: Figure S5). In PARP1, an additional N-terminal β-strand forms a three-stranded antiparallel β-sheet with the primary β-hairpin of the treble clef and connects to the zinc-knuckle containing β-hairpin, forming a topologically similar connection to that seen in the UBR-box.
We show that the UBR-box is a novel member of the RING-like treble clef fold and is related to the B-box and ZZ domains. The UBR-box is present only in eukaryotes and appears to have emerged during the expansion of ubiquitin system pathway-related functions [48, 9]. However, its function is not merely confined to recognition of N-recognins and it may serve diverse biological roles by mediating interactions with other proteins. The structural modifications including the circular permutation and the functionally important extensions to the RING-like core has resulted in the emergence of a novel UBR-box fold capable of binding type 1 N-degrons. The relationship of RING-like domains to the UBR-box, illustrates the versatility of the treble clef scaffold upon which novel functions may emerge on different regions of the domain [8–10, 55].
Structure based methods
Structures of the human and S. cerevisiae UBR-box (PDB identifiers 3NY1, 3NY2, 3NY3, 3NIH, 3NII, 3NIJ, 3NIK, 3NIL, 3NIM, 3NIN, 3NIS and 3NIT) and other treble clef domains were retrieved from the PDB. Dali , TopSearch , TM-align  and Fr-TM-align  tools were used to evaluate structural similarity of the UBR-box with other proteins. The structures were visualized, compared and manually superimposed in the molecular visualization program, PyMOL. Manual structural superimposition was performed by defining the equivalent regions using the pair fitting command of PyMOL.
Sequence based methods
Sequence similarity searches initiated with the UBR-box domain were performed using iterative PSI-BLAST (against PDB database of Feb 28, 2015, Number of letters: 18,460,040, Number of sequences: 75,710; E-value threshold of 0.001) , JackHMMER program from the HMMER3 package (against NCBI non-redundant (NR) version 2014-06-17 and UniProtKB version 2014-06-17; E-value threshold of 0.01) , FFAS server  (against regularly updated PDB, Pfam and SCOP databases) and HHpred server  (against PDB70_ 24Jan15 and PfamA_27.0, using MSA generation method HHblits run for 5 iterations, E-value threshold of 0.001). MSAs of the treble clef domains were made using the ClustalW program  within the BioEdit software package (version 7.2.2) with default parameters . Thereafter, structure based manual adjustment of the MSAs was done.
Review #1: Eugene V. Koonin, Senior Investigator, National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD USA
Report form: Kaur and Subramanian examine the structure of the UBR box and show that it is a circular permutation of the binuclear treble clef Zn finger rather than a truly novel Zn-binding fold. To the best of my understanding, this is a valid relationship and as such, quite a useful observation. In my opinion, the authors miss out on phylogenomic analysis of the UBR box. I think it would have been quite informative to identify UBR box-containing proteins across the entire eukaryotic diversity and reconstruct the scenario of evolution. Is N-degron recognition the only function of the UBR box or are there additional exaptations? This question could be addressed through careful analysis of the domain architectures of the UBR box-containing proteins. As it stands, the manuscript reads more like a Discovery Note although the format is that of a research article. Expansion along the above lines could rectify this mismatch between content and form.
The manuscript is not carefully written or edited, careful editing is a must.
Quality of written English: Needs some language corrections before being published
Response to the Report:
We have now analysed and reconstructed phylogeny for UBR-box domains across all eukaryotes. A phylogenetic tree of representative UBR-box domains is included as an additional figure. Domains co-occurring with the UBR-box are provided besides the branch labels. Our analysis, similar to earlier studies, reveals that the UBR-box co-occurs with a diverse set of protein domains. Most domain architectures are suggestive of a likely involvement of the UBR-box containing proteins in ubiquitin-related pathways. However, it is also known that many UBR-box containing proteins lack the N-recognin function and do not bind N-degrons. In such scenarios, the most plausible function of the UBR-box may be in mediating protein-protein interactions, as discussed in the manuscript.
Review #2: Dr. Balaji Santhanam, Senior Investigator Scientist, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK
There seems to some intertwined arguments for relationship between UBR-box and binuclear treble clef fingers. These include systematic structural searches, followed by sequence searches and also manual analyses (Line # 107 to 152). The organization and writing of this part is confounding the understanding of the strategies to link UBR to binuclear treble clef fingers.
I found it rather strange that the authors narrate their structural analyses first and discussed the sequence analyses later. Sequence searches clearly show clear link between UBR and ZZ, B-Box (at least for significant parts of the domains). This also possibly adds to the confusion stated above. I would have naturally expected sequence searches to be the first straightforward way to get an evolutionary link between domains. Hence, has to be the first one to be discussed here.
In response to 1 and 2:
There was no detailed discussion on their observation of evolutionary link between ZZ, B-box and UBR later as well.
Given there is strong sequence/structure relationship between UBR-box and ZZ and B-box, I was expecting an evolutionary model similar to the Ref. 9 (Burroughs et al.). Ref. 9 suggests that FYVE finger is likely to have evolved from a mononuclear version through LIM like intermediate through circular permutations (CP). Is it possible that UBR-box evolved by CP of either ZZ or B-box precursor? Did the authors look for UBR-like proteins in prokaryotes?
In response to 3 and 4,
As articulated in Ref. 9 binuclear treble clef fingers have independently or by convergence acquired Ub-related functions. UBR seems to be no exception; do the authors have any comment?
Structural search strategy appears less systematic than the sequence analyses. I understand that this is possibly due to DALI searches did not yield any significant hits. Did the authors try running DALI locally on an expanded database of structures?
It is rather strange to me that structural searches using TopSearch yielded best hit to PARP1 and not to ZZ or B-box as suggested by sequence searches. Do the authors have any explanation?
TopSearch perhaps finds highest similarity to PARP1 because of an extensive structural similarity of the protein domains, especially in the regions involved in ligand binding. We notice that the UBR-box and the treble clef domain of PARP1 have a similar twist in their respective primary β-hairpins. Also, the additional β-strand which forms a three stranded β-sheet with the primary β-hairpins in both the proteins is similarly extended into the knuckle-containing β-strand and helps sculpt the respective ligand binding regions.
Review #3: Kira S. Makarova, National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD USA
Report form: The paper presents a relatively straightforward sequence and structure comparative analysis of several families of RING treble clef zinc finger domain. Based on this analysis authors hypothesized that UBR box domain originated though a circular permutation of the ancestral RING-like domain. This hypothesis considering presented data appears plausible and the text and figures are generally clear. I don’t have any significant criticism for this work. The only problem I have with the paper is its length and relevance of some parts of the text and some Figures. I believe that the arguments in support of this hypothesis could be described in a text half of the current size and rather in a format of a Discovery Note paper than the original research paper.
For a better evolutionary perspective and understanding the UBR-box family I’d recommend to start Result and Discussion with a brief description of diversity of this domain in different eukaryotic lineage. It can be described together with results of other sequence similarity searches and the Fig. four should be moved forward accordingly. Furthermore the details of sequence similarity searches could be moved to Additional material and only essence of that searches mentioned in the text. In the Fig. four organisms to which aligned sequences belong should be indicated and more UBR-box sequences from different eukaryotic lineages should be shown in the multiple alignment accordingly. Otherwise it not clear when the permutation happened during the evolution of this domain.
We have taken all suggestions of the reviewer to improve the manuscript. We have added details relating to the distribution of the UBR-domain in eukaryotes in the Results and discussion section.
There is no need to mentioned other cases of the permuted treble clef zinc finger domain, especially if they are not described as such in the literature (for example, domain in CasA protein of CRISPR-Cas system and respective Additional material). So if authors find it interesting then they should write a different a paper showing this explicitly or a review describing this phenomenon in general.
Examples of circular permutations in the treble clef domain are rather rare. We have only mentioned well documented cases of circular permutations with the exception of the CRISPR system Cascade subunit CasA. The purpose of this section was to illustrate that the circular permutation observed in UBR-box is not unprecedented and a similar permutation is observed in the third treble clef domain of the triquetra knot containing protein Rds3p. For this reason, we would like to retain this section.
On the Fig. 2 a please indicate respective cysteines and histidines for Zn ligands. Indicate the fact that pink strands are actually not structured in UBR-box structures.
The zinc-chelating residues have not been labelled in Fig. 2a as there isn’t absolute conservation of the aminoacids involved in metal-chelation. Cysteines and histidines are the most commonly observed residues which chelate the zinc ions as is evident from the MSA.
The representation of the UBR-box in Fig. 2a is shown with the β-strands colored in pink to indicate equivalencies among the various RING-like domains. Although, the pink colored β-strands of the UBR-box are not structured in the apo-form, at least one of these takes up a β-strand conformation in the holo-form. These structural details are also mentioned in the text.
Comparison with PARP1 mononuclear treble clef does not add anything to the story, as the Fig. 5. It can be either moved to the supplement or omitted entirely. The same applies to the Fig. 3 .
We have moved these figures to the Additional files section as suggested by the reviewer. However, we would like to retain the text which compares PARP1 and the UBR-box. PARP1 is among the best scoring hits in TopSearch and binds DNA in a similar region to that used by UBR-box to bind the N-degron peptide. Analysis of binding sites in all treble clef domains suggest that this region is unique to these two proteins and we feel it is appropriate to discuss this in the context of the UBR-box.
Reviewers’ comments – second round of review
Reviewer #1 and #2 had no additional comments.
Report form: The paper presents a relatively straightforward sequence and structure comparative analysis of several families of RING treble clef zinc finger domain. Based on this analysis authors hypothesized that UBR box domain originated though a circular permutation of the ancestral RING-like domain. This hypothesis considering presented data appears plausible and the text and figures are generally clear. I don’t have any significant criticism for this work.
After the first round of revision the clarity of the paper improved, although there are still some parts of the paper that are not explained in sufficient details and are not directly relevant for the main point of the paper. As I mentioned before this concerns circular permutations in the treble clef domain of CasA protein and the triquetra knot in Rds3p protein which were “noticed” but not elaborated by the authors (now both are shown in the supplement). Since no analysis of these cases were provided this leaves a reader with the only option to believe that the authors are correct in their interpretations.
Taking into consideration the concerns of the reviewer, we have removed the text and figure illustrating the circularly permuted treble clef motif which we observed in the CasA protein. However, we retain the prolyl-tRNA synthetase and Rds3p examples as these treble clef motifs have already been documented and discussed in previously available literature [10, 47].
I also still have an issue with the authors’ response to my request to indicate aminoacids involved in Zn binding on the Fig. 2a in order to have a better understanding of the amino acid signatures of the 1st and 2nd Zn binding motif. I expect that each family shown in the Fig. 2a has a typical signature (consensus) for Zn binding ligands. These residues don’t have to be 100 % conserved. Showing this consensus is the same as showing a generalized representation of the secondary structure elements for the respective domains (which is shown in the Fig. 2a ), although some proteins could have deviations from this typical organization.
We have added the metal-chelating aminoacids for the domains as per Pfam consensus in Fig. 2a and also provide a short description in the figure legend. The side chains of the metal-chelating residues are also shown as sticks for each of the domains in their tertiary structures in Fig. 2b-f.
ubiquitin ligase N-recognin
Really Interesting New Gene
Protein Data Bank
Structural Classification of Proteins
Class, Architecture, Topology, Homology
Evolutionary Classification of protein Domains
Multiple Sequence Alignment
Last Eukaryotic Common Ancestor
Last Universal Common Ancestor
Poly (ADP-ribose) polymerase
Homologous to the E6-AP Carboxyl Terminus
This work was supported in part by the XII Five-year plan network project GENESIS (BSC0121) and intramural funds (OLP_0072) from the Council of Scientific and Industrial Research (CSIR) - Institute of Microbial Technology, Chandigarh, India. G.K. is supported by the Shyama Prasad Mukherjee Fellowship of CSIR, India. The authors are grateful to T.N.C. Ramya for critical reading of the manuscript and Indu Khatri for helpful comments.
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