While the initial reaction catalyzed by Thg1 has been compared to adenylation catalyzed by the aminoacyl tRNA synthetases, the evolutionary relationships of Thg1 and the actual nature of the polymerase reaction catalyzed by it remain unclear.Almost all known nucleic acid polymerases catalyze 5'-3' polymerization by mediating the attack on an incoming nucleotide 5' triphosphate by the 3'OH from the growing polynucleotide chain in a template dependent or independent manner. The only known exception to this rule is the Thg1 RNA polymerase that catalyzes 3'-5' polymerization ++ cations. Based on this we predict that Thg1 catalyzes polymerization similarly to the 5'-3' polymerases, but uses the incoming 3' OH to attack the 5' triphosphate generated at the end of the elongating polynucleotide. In addition we identify a distinct set of residues unique to Thg1 that we predict as comprising a second active site, which catalyzes the initial adenylation reaction to prime 3'-5' polymerization. Based on contextual information from conserved gene neighborhoods we show that Thg1 might function in conjunction with a polynucleotide kinase that generates an initial 5' phosphate substrate for it at the end of a RNA molecule. In addition to histidinyl tRNA maturation, Thg1 might have other RNA repair roles in representatives from all the three superkingdoms of life as well as certain large DNA viruses. We also present evidence that among the polymerase-like domains Thg1 is most closely related to the catalytic domains of the GGDEF and CRISPR polymerase proteins.Using sensitive profile-profile comparison and structure prediction methods we show that the catalytic domain Thg1 contains a RRM (ferredoxin) fold palm domain, just like the viral RNA-dependent RNA polymerases, reverse transcriptases, family A and B DNA polymerases, adenylyl cyclases, diguanylate cyclases (GGDEF domain) and the predicted polymerase of the CRISPR system. We show just as in these polymerases, Thg1 possesses an active site with three acidic residues that chelate MgBased on this relationship and the phyletic patterns of these enzymes we infer that the Thg1 protein is likely to represent an archaeo-eukaryotic branch of the same clade of proteins that gave rise to the mobile CRISPR polymerases and in bacteria spawned the GGDEF domains. Thg1 is likely to be close to the ancestral version of this family of enzymes that might have played a role in RNA repair in the last universal common ancestor.This article was reviewed by S. Balaji and V.V. Dolja. Nucleic acid polymerase activity has emerged independently in at least four structurally unrelated folds, namely the RNA-recognition motif (RRM) fold , the double ψ-beta barrel, the polβ-like and TOPRIM folds -3. Yet, In the case of the template-independent ligation of G-1 the reaction proceeds via an initial adenylation of the 5' phosphate by Thg1 resulting in a 5' App overhang . This isWe were interested in the unusual catalytic properties of Thg1 and were intrigued by its mysterious evolutionary affinities. As a part of our effort to understand the origins of nucleic acid polymerization, we investigated Thg1 using sensitive sequence-profile methods and showed that it belongs to the RRM-fold nucleic acid polymerase palm domains. Furthermore, we show that it is most closely related to the catalytic domains of the diguanylate cyclases (GGDEF) and CRISPR system polymerases and provides new information regarding the origin and functions of these domains.Pyrobaculum aerophilum Thg1 recovered GGDEF proteins from Hahella chejuensis . Although, these hits had only marginal significance, they perfectly aligned with the three principal acidic catalytic residues of the diguanylate cyclases/GGDEF domain  with conditional e-values as low as 6.1 × 10-5. We then predicted secondary structure using the Jpred program [3ign). The predicted secondary structure of Thg1 showed a conserved core of 4 strands and two helices . Additional profile-profile searches with an in-house group of profiles, which includes domains not properly represented in PFAM, recovered the catalytic domain of the polymerases of the CRISPR system [To investigate the affinities of the Thg1 polymerase, we initiated sequence profile searches using the PSI-BLAST program  with sevR system . This st2+ ions that are central to the catalytic mechanism of these enzymes. A common mechanism is deployed irrespective of whether an enzyme of this family catalyzes elongation of a polynucleotide chain or nucleotide cyclization  or a second nucleotide (polymerization and cyclic diguanylate generation). A comparison of the Thg1 catalytic core with that of the palm domain from the above enzymes shows that it shares with the GGDEF, CRISPR, viral RdRP and RT the configuration of the active site at the hairpin, i.e. two acidic residues preceded by a small residue  RNA-dependent RNA polymerases of positive strand RNA viruses and their derivatives ; 2) reverse transcriptases (RT); 3) DNA polymerase family B, including the archaeo-eukaryotic replicative enzymes; 4) the DinB-like DNA-repair polymerases; 5) DNA polymerase family A; 6) the phage T7-type DNA polymerases; 7) the phage T7-type DNA-dependent RNA polymerases; 8) the TV-polymerase family; 9) the CRISPR-system polymerases; 10) GGDEF domains; 11) nucleotide cyclases (cAMP and cGMP generating enzymes) . The cat2+-dependent catalytic mechanism as in case of the other polymerase palm domains  . MACRO dTrichomonas vaginalis, which is believed to be an early-branching eukaryote. Further, within the eukaryotes, monophyletic lineages such as animals, plants, apicomplexans and ciliates can be discerned suggesting that Thg1 was present in the common ancestor of all extant eukaryotes ; HEVC: Human enterovirus C; Haur: Herpetosiphon aurantiacus; HIV1: Human immunodeficiency virus 1; Hsap: Homo sapiens; Lbic: Laccaria bicolor; Mace: Methanosarcina acetivorans; Mbar: Methanosarcina barkeri; Mbre: Monosiga brevicollis; Mbur: Methanococcoides burtonii; Mcap: Methylococcus capsulatus; Mcht: Microcoleus chthonoplastes; Mgam: marine gamma; Mhun: Methanospirillum hungatei; Mjan: Methanocaldococcus jannaschii; Mkan: Methanopyrus kandleri; Mmar: Microscilla marina; MMLV: Moloney murine leukemia virus; Msmi: Methanobrevibacter smithii; Msta: Methanosphaera stadtmanae; Mthe: Methanothermobacter thermautotrophicus; Mxan: Myxococcus xanthus; Ncra: Neurospora crassa; Nelo: Neisseria elongata; Nfar: Nocardia farcinica; Nvec: Nematostella vectensis; Pfal: Plasmodium falciparum; Plim: Planctomyces limnophilus; Ppac: Plesiocystis pacifica; Ppat: Physcomitrella patens; Ppla: Postia placenta; Psyr: Pseudomonas syringae; Ptet: Paramecium tetraurelia; Pyae: Pyrobaculum aerophilum; S2RNV: Saccharomyces 23S RNA narnavirus; Scel: Sorangium cellulosum; Scer: Saccharomyces cerevisiae; Spom: Schizosaccharomyces pombe; Ssol: Sulfolobus solfataricus; Sulf.: Sulfitobacter sp.; Ster: Sebaldella termitidis; Taqu: Thermus aquaticus; Tbru: Trypanosoma brucei; Tneu: Thermoproteus neutrophilus; Tthe: Tetrahymena thermophila; Tvag: Trichomonas vaginalis; Tvol: Thermoplasma volcanium; Vspi: Verrucomicrobium spinosu.The authors declare that they have no competing interests.VA, LMI and LA performed the reported research and wrote the paper. All authors read and approved the final manuscript.Balaji Santhanam, Center for Cancer Systems Biology, Dana-Farber Cancer InstituteDepartment of Genetics, Harvard Medical School, Boston MAIn this manuscript titled "Presence of a classical RRM-fold palm domain in Thg1-type 3'- 5'nucleic acid polymerases and the origin of the GGDEF and CRISPR polymerase domains" Anantharaman et al report the computational discovery of a RRM-fold domain in Thg1 family proteins and its evolutionary relationship to bacterial GGDEF domains and CRISPR polymerases, which are predominantly found in archaea. The authors also provide detailed descriptions of their catalytic sites and functional contexts based on amino-acid residue conservations, domain architectures and protein interaction information. Further, the authors discuss the common evolutionary origins of Thg1, GGDEF and CRISPR polymerase families and propose a biochemical role for the RRM-fold palm domain in LUCA. The manuscript reports fascinating findings and would aid future experimental investigations along these lines. I strongly support the publication of the manuscript in Biology Direct.Few points to the authors:1. Do all Thg1 family proteins have secondary catalytic site or is there any exception? If there is one, in that specific case, can the authors comment on how the adenylation process would be coupled to 3'-5' polymerization?As indicated above the Thg1 orthologs from seed plants have a duplication of the Thg1 polymerase domain. The N-terminal domain lacks the DxR motif associated with secondary active site. We propose that in this case there could be a "division of labor" with the N-terminal domain mediating chain elongation but the C-terminal domain capable of adenylation as well as polymerization.2. Do the authors find the N-terminal helical extension region to the core RRM domain present in most of Thg1 family proteins? If it is true, do the authors think there is any potential role for this region in directing the specificity for 3'-5' polymerization?The N-terminal helix is present in all the Thg1 proteins. It is certainly functionally important because mutation of a glutamate in this helix reduces activity. However, there are no absolutely conserved residues suggesting that it is unlikely to contribute directly to the active site. It could instead play a role in dimerization.3.Does the C-terminal HTH-like region of Thg1 have roles other than contributing to active-site biochemistry, like mediating interactions with nucleic acids or proteins?It could interact with the nucleic acid template comparable to the "fingers" module of other nucleic acid polymerases. However, beyond this there is no available evidence that it mediates other interactions.4.Thg1p of budding yeast seems to form a homodimer based on protein interaction data from two independent experiments. Would this information be helpful in providing additional insights into the functional contexts of Thg1 family? The seed plant versions contain two distinct Thg1 domains that might show functional differentiation (see above). This observation, together with evidence from yeast for homodimerization, suggests that the Thg1 protein is indeed likely to function as a dimer. It remains to be seen if this dimerization might imply partitioning of adenylation and polymerization between the two monomers in the functional unit.5.Thg1p of budding yeast seems to be associated with Orc2p directly in a complex. Again based on the protein- and genetic- interaction data Thg1 (in budding yeast) seems to be associated with proteins/genes involved in DNA replication/repair. Is it possible that the Thg1 family proteins could function in the context of DNA replication/repair as well?This is indeed an intriguing observation - given that Thg1 shows both physical and genetic interactions with Orc2 it is likely that this association is functionally relevant. Of the other genetic interactions of Thg1 there are multiple interactions with genes involved in tRNA- or translation- related functions such as Hts1: the histidinyl tRNA synthetase which utilizes the HtRNA produced by Thg1 as a substrate, Trm11: the tRNA methylase which methylates G10 of tRNAs, and Tma64: a protein with a RNA-binding Sui1 domain involved in RNA processing. These are consistent with the role of Thg1 in HtRNA maturation. However, in addition to Orc2, Thg1 shows genetic interactions with other DNA replication related genes such as Pol32: a winged HTH containing subunit of the DNA polymerase delta required for replication and error prone repair, and Mrc1: A regulator of replication and repair. This suggests that role of Thg1 in DNA replication might indeed be relevant. While there is currently no biochemical evidence that throws light on this role, it would be of interest to investigate if its adenylation or polymerization activity might have a direct role in processes such as priming and ligation.6.The authors could indicate catalytic residues explicitly  in the figure This would greatly aid in comprehending the relevant parts of the manuscript.The proposed catalytic residues have been indicated by means in blue boxes in Fig. Valerian V. Dolja, Department of Botany and Plant Pathology, Oregon State UniversityCordley Hall 2082, Corvallis, ORThis incisive study from Aravind's shop uses cutting-edge bioinformatics analyses to add yet another colorful piece to a jigsaw puzzle of the early life origins and evolution. It starts with a relatively obscure albeit mechanistically enigmatic class of Thg1 enzymes involved in maturation of a single tRNA, tRNAHis. Unexpectedly, it uncovers relationships of Thg1 with GGDEF-like catalytic domains, CRISPR system polymerases that are very much in vogue, and, finally, connects them to a vast class of RRM-fold palm domain polymerases.Being a virologist turned cell biologist, I am not in position to comment on the intricacies of the methodology used to unveil structural, mechanistic and phylogenetic relationships of Thg1; I simply trust the authors by virtue of their impressive record. Rather, I would like to emphasize the most intriguing implications of their work. First, their study suggests likely catalytic mechanisms for both Thg1 and CRISPR polymerases that are experimentally testable. Second, it divines the evolutionary scenario for Thg1, GGDEF domains, and CRISPR polymerases that, although not immediately testable, proposes a feasible and broad RNA repair role for their common ancestor. This possibility, in turn, strengthens the argument for primordial RNA-based genetic systems allowing for more faithful RNA replication and larger RNA-based genomes. In addition, it stimulates further inquiry into extant RNA repair systems, a relatively young research area that recently uncovered RNA repair roles for the cellular and viral AlkB demethylases [thylases .Aas, P.A., Otterlei, M., Falnes, P.O., Vagbo, C.B., Skorpen, F., Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E., and Krokan, H.E. (2003). Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421, 859-863.van den Born, E., Omelchenko, M.V., Bekkelund, A., Leihne, V., Koonin, E.V., Dolja, V.V., and Falnes, P.O. (2008). Viral AlkB proteins repair RNA damage by oxidative demethylation. Nucleic Acids Res. 36, 5451-5461.I have only two more specific and indeed minor comments.1. It would be helpful to include at least a schematic dendrogram depicting results of phylogenetic analysis (now shown in Addit. File 1) as a panel in a figure.We include a schematic of the phylogenetic tree shown in Fig.2. On page 8, both mimivirus and caudovirus are collectively called 'phages'. Traditionally, this term reserved for viruses of bacteria only, whereas mimiviruses infect unicellular eukaryotes.We alter this to refer to only the caudovirus as a phage.RRM-fold palm domain in Thg1-type 3'- 5'nucleic acid polymerases. Complete phyletic pattern, domain architectures and alignment data along with more detailed material and methods are provided in this file. The file can also be accessed from: ftp://ftp.ncbi.nih.gov/pub/aravind/Thg1/Thg1.html.Click here for file


Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 Å resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.Synaptotagmins are known to mediate diverse forms of Ca Exocytosis at different types of secretory cells is governed by similar protein machineries that are fine tuned for specific regulatory requirements 2+ sensors and are believed to underlie at least in part the distinct Ca2+ dependencies of different forms of regulated secretion 2+ sensor that triggers fast neurotransmitter release at the hippocampus 2+-dependent secretion is mediated by synaptotagmins-1 and -9 2+ sensors The release of hormones and neurotransmitters by Ca2 domains that form most of their cytoplasmic regions (referred to as C2A and C2B domains). In this context, the C2 domains of synaptotagmin-1 have been the subjects of particularly extensive study. Both domains form characteristic β-sandwich structures that bind three or two Ca2+ ions  through loops located at the top of the β-sandwich, and do not undergo substantial conformational changes upon Ca2+ binding 2B domain contains two α-helices that are not present in the C2A domain; one of these helices (HA) is generally found in C2 domains of tandem C2 domain-proteins 2 domains bind to phospholipids in a Ca2+-dependent manner through the Ca2+-binding loops at the top face 2+ affinity of this activity lead to parallel changes in the Ca2+ dependence of neurotransmitter release 2+ binding to the C2B domain is much more critical for release than Ca2+ binding to the C2A domain 2B domain (but not the C2A domain) can bind to membranes through the bottom face, which allows simultaneous binding to two closely apposed membranes and suggests that synaptotagmin-1 may trigger neurotransmitter release by bringing the synaptic vesicle and plasma membranes together in a Ca2+-dependent manner 2B domain practically abolishes synaptotagmin-1 function 2B domain Functional differentiation among synaptotagmins is expected to arise primarily from differences in the properties of the two C2 domains and those from other synaptotagmin isoforms and other C2 domain-containing proteins involved in membrane traffic, it seems likely that many of these C2 domains share at least some of the properties found in synaptotagmin-1 – see 2 domains of some of these proteins lack a full complement of predicted Ca2+ ligands and, as a consequence, do not bind Ca2+ or phospholipids; instead they act as protein-protein interaction domains [e.g. the Munc13-1 C2A domain 2 domains with high sequence similarity with the synaptotagmin-1 C2 domains can have dramatically different properties. For instance, the piccolo/aczonin C2A domain exhibits a dramatic Ca2+-dependent conformational change 2B domain does not bind Ca2+, despite having all the predicted Ca2+-binding ligands, because of conformational differences with respect to the synaptotagmin-1 C2B domain in the Ca2+-binding region 2 domains based on sequence analyses alone, and the basis for functional differences among synaptotagmins is poorly understood. The relation between the functions of synaptotagmins-1 and-7 is particularly fascinating, as data obtained in chromaffin cells 2+ sensor for synaptic vesicle exocytosis despite being enriched in synapses synaptotagmin-1 knockout (KO) mice, unlike synaptotagmins-2 and -9 Based on the sequence similarities between the synaptotagmin-1 C2B domain replaced by the synaptotagmin-7 C2B domain, is also unable to rescue fast neurotransmitter release in synaptotagmin-1 KO neurons. Even extensive mutagenesis of this chimera to make the C2B domain more similar to that of synaptotagmin-1 did not lead to rescue of fast release. Furthermore, deletion of the HB helix in synaptotagmin-1 led to a gain of function, showing that the absence of this helix in synaptotagmin-7 does not underlie its inability to rescue fast release. We describe the crystal structure of the Ca2+-bound synaptotagmin-7 C2B domain at 1.44 Å resolution, which is very similar to that of the synaptotagmin-1 C2B domain. The synaptotagmin-7 C2B domain structure also shows that this domain binds three Ca2+ ions and that coordination of two of the Ca2+ ions is analogous to that found previously in solution by NMR spectroscopy for the synaptotagmin-1 C2B domain 2+ coordination observed in a crystal structure of the latter 2B domain for synaptotagmin-1 function. Although we cannot completely rule out the possibility that the lack of rescue of fast release by the Syt1/7 chimeras might be due to problems with proper targeting to synaptic vesicles and/or protein folding, our data suggest that the functional differences between the synaptotagmin-1 and –7 C2B domains do not arise from conformational differences but rather from subtle alterations in biochemical properties caused by multiple residue substitutions. These results also illustrate how it is much more difficult to confer a specific functional activity of a protein to a closely related isoform than to disrupt this activity.In this paper, we describe attempts to shed light onto this apparent paradox through a combination of functional and structural studies. We find that a chimeric protein containing the synaptotagmin-1 sequence, but with the C2+ binding to the C2B domain is more critical than Ca2+ binding to the C2A domain for synaptotagmin-1 function 2B domain with the synaptotagmin-7 C2B domain allows or abolishes rescue. For this purpose, we constructed a lentiviral vector expressing a chimeric protein that contains most of the synaptotagmin-1 sequence, but with the C2B domain replaced by the synaptotagmin-7 C2B domain  and an R399Q mutation strongly impairs synaptotagmin-1 function 2B domain and is asparagine (N396) in synaptotagmin-1; A295, which is at the top of the C2B domain and is lysine (K301) in synaptotagmin-1; and E318, which is in the middle of a characteristic polybasic region at the side of the C2B domain and is lysine (K324) in synaptotagmin-1. We also replaced two loops at the bottom of the C2B domain , as well as the linker region between the C2A and C2B domains  (2+-triggered neurotransmitter release in synaptotagmin-1 KO neurons (2B domains.A summary of the mutants of the Syt1/7 chimera that we generated is shown in agmin-1) . Immunocagmin-1) . However neurons . It is i2B domain showed that a major structural difference between the C2A and C2B domains of tandem C2 domain proteins is the presence of helix HA at the bottom of the β-sandwich 2B domain contains an additional helix at the C-terminus (helix HB) that is predicted to be present only in synaptotagmins-1, -2 and -8 2B domain could explain its inability to rescue release in synaptotagmin-1 KO neurons. To test this hypothesis, we prepared a lentiviral vector expressing a synaptotagmin-1 fragment lacking the HB helix  and performed additional rescue experiments in synaptotagmin-1 KO neurons. Western blotting and immunocytochemistry showed that Syt1 delta HB is expressed at similar levels as WT synaptotagmin-1 and properly targets to presynaptic terminals in synaptotagmin-1 KO neurons  and the synaptotagmin-1 C2B domain structures obtained by X-ray (orange) or NMR (yellow) might be meaningful, since the conformations of these loops in the two synaptotagmin-1 C2B domain structures are clearly more similar to each other than to their conformations in the synaptotagmin-7 C2B domain. However, loops 1 and 3 are known to have some degree of flexibility in C2 domains due to their exposed nature, and they are involved in extensive crystal contacts for the synaptotagmin-7 C2B domain  and is not physiologically relevant.The presence of Ca2+ ions bound at the cavity formed by the top loops are coordinated by five aspartate and one serine side chains, as well as by three backbone carbonyl groups (2+-binding sites are numbered 1–3 as is common for C2 domains 2A domain 2B domain of synaptotagmin-1 was found to bind only two Ca2+ ions 2+ ion in the synaptotagmin-1 C2A domain (S235) and the synaptotagmin-7 C2B domain (S362). Note that this serine residue of loop 3 is not generally conserved in synaptotagmin C2B domains  from a neighboring molecule in this structure  also abolished synaptotagmin-1 function, showing that not only the top, Ca2+-dependent face, but also the bottom, Ca2+-independent face of the C2B domain plays a key role in release 2+ ligands at the top loops and the two arginines at the bottom face of the C2B domain, and yet they are unable to rescue neurotransmitter release in synaptotagmin-1 KO neurons. It is important to note that the inability of at least some of the Syt1/7 chimeras to rescue fast release might arise because of improper protein targeting and/or folding. However, our immunocytochemistry data show that the chimeras were targeted to synapses, and previous results with different synaptotagmin-1/7 chimeras showed that including the N-terminal sequence of synaptotagmin-1 that contains an N-glycosylation site is sufficient for targeting to vesicles 2+-binding sites at the top face and the arginines at the bottom face of the C2B domain are necessary but not sufficient for synaptotagmin-1 like function.First, our data confirm and extend previous results that revealed the central importance of the C2B domain, which was recently proposed to be important for synaptotagmin-1 function 2+, phospholipids and SNAREs 2B domain seems to be involved in SNARE binding 2B domain contains a Lys to Glu substitution in this region (E318), perhaps this substitution leads to weaker SNARE binding. However, no substantial differences between synaptotagmins-1 and -7 in SNARE binding were observed in a previous comparative study 2+, phospholipid and SNARE binding might not be sufficient for synaptotagmin-1 function. Clearly, further research will be necessary to test this possibility. For instance, synaptotagmin-1 appears to bind to the SNAREs in diverse modes, including an interaction with the syntaxin-1/SNAP-25 heterodimer that promotes vesicle docking A potential explanation for the inability of synaptotagmin-7 2B domain back to those present in synaptotagmin-1 (mutant #9), since the resulting C2B domain is highly similar to the synaptotagmin-1 C2B domain and contains all the residues that have been identified so far to be critical for synaptotamin-1 function. Moreover, the crystal structure of the synaptotagmin-7 C2B domain described here shows that its backbone structure and even the conformations of many side chains are very similar to those of the synaptotagmin-1 C2B domain, suggesting that their functional differences do not arise from distinct structural properties. In contrast, the synaptotagmin-4 C2B domain was indeed found to have marked conformational differences with respect to the synaptotagmin-1 C2B domain that explained the inability of the former to bind Ca2+2B domain has a full complement of Ca2+ ligands (2B domains of synaptotagmins-1 and -4 have a similar degree of sequence identity (45%) to that between the C2B domains of synaptotagmins-1 and -7 (48%). All of these observations show that, although sequence analyses are sometimes very useful to predict biochemical and functional properties of proteins, such properties are often very difficult to predict and rationalize from the sequences alone. It is tempting to speculate that subtle sequence and/or structural distinctions among isoforms might be key to dictate dramatic functional differences.Although we were unable to achieve rescue of fast neurotransmitter release in synaptotagmin-1 KO neurons with the Syt1/7 chimeras, our experiments with multiple mutants underscore how difficult it is to confer a specific activity to a protein isoform even when the starting point is a closely related isoform. Although this conclusion might seem trivial, it has not been sufficiently appreciated in the past to the best of our knowledge. Note that no rescue was observed even when we restored 30 residues of the chimera C ligands , and tha2B domain described here and that of the synaptotagmin-1 C2B domain  does not directly coordinate Ca2+2B domain cannot determine Ca2+-binding modes accurately 2+, as implied in ref. 2+-binding modes of both synaptotagmin-1 C2 domains determined with our NMR methodology 2 domains , respectively. The same vector without synaptotagmin served as a control construct. The cDNA of wildtype mouse synaptotagmin-1 was cloned into the lentiviral vector and was used to generate synaptotagmin-1 delta HB mutant by standard recombinant DNA techniques. The cDNAs of wildtype rat synaptotagmin-1 and synaptotagmin-7 were used to generate chimeric synapotagmin-1/7 variants, which were subsequently cloned into the lentiviral vector. Lentiviruses were produced by co-transfecting HEK 293T cells with the lentiviral vector and two helper vectors, pVSVg and pCMV-delta R8.9 vector A modified lentiviral vector Synaptotagmin-1 knockout mice were obtained by interbreeding of mice heterozygous for the synaptotagmin-1 mutation as described −2 on WT astrocyte microislands to obtain autaptic neurons for electrophysiology and immunocytochemistry experiments. Neurons were plated at the density of 10,000 cm−2 on confluent WT astrocytes for Western blot experiments.Primary neuronal cultures were prepared as described in vitro 10 were fixed with 4% (w/v) paraformaldehyde for 30 minutes at room temperature and the following primary antibodies  were used: rabbit anti-GFP (1∶1000), guinea pig anti-vesicular glutamate transporter-1 (1∶1000), rabbit anti-synaptophyin-1 (1∶1000), and mouse anti-synaptotagmin-1 . Images were acquired on a Leica TCS SP5 laser-scanning confocal microscope or a Zeiss 510 laser scanning confocal microscope. The settings for laser power and detector gain allowed the pixel intensities to remain within the dynamic range. Images were acquired as z-stacks with 8-bit and 1024×1024 pixel resolution and processed using software NIH ImageJ 1.40 g to create sum projections from the stacks.Immunocytochemistry was performed as described Western blotting was performed as described 2, 4 : KCl, 136; HEPES, 17.8; EGTA, 1; MgCl2, 0.6; ATP-Mg, 4; GTP-Na, 0.3; Phosphocreatine, 12; Phosphocreatine kinase, 50U ml−1; 300 mOsm; pH 7.4. Action potential-evoked EPSC, readily releasable pool size, vesicular release probability, short-term plasticity, and apparent Ca2+ sensitivity of release were measured as described t-test, one-way analysis of variance, or two-way analysis of variance.Whole-cell voltage clamp experiments were performed as described 2, 4 for  or 2 , cleavage from the GST moiety and purification were performed as described previously for the synaptotagmin-1 C2B domain 2B domain was concentrated to 40 mg/ml in 25 mM NaAcetate buffer pH 6.2, 50 mM KCl, 20 mM CaCl2, and 1 mM TCEP. After concentration, a UV spectrum was recorded to determine protein concentration and ensure that the protein was not contaminated by DNA/RNA The vector to express the mouse synaptotagmin-7 C2B domain structure (PDB accession code 1TJM) as a search model. 136 of 138 residues were automatically built into the electron density and the sequence was assigned by the program ARP/warp 2B domain contains residues 266––403, 4 Ca2+ ions and 183 waters . For data collection and refinement statistics, see The crystallographic data were indexed and integrated using HKL3000 Figure S1Delta HB mutant synaptotagmin-1 is expressed at similar levels as WT synaptotagmin-1 and properly targets to presynaptic terminals. synaptotagmin-1 KO neurons were infected with lentiviruses expressing EGFP alone or Syt1 and EGFP together. (A) Exemplary Western blot showing similar expression levels of WT and delta HB Syt1. Neuron specific b-Tubulin III serves as loading controls. (B) Exemplary confocal images showing the presynaptic localization of WT and delta HB Syt1. Neurons were immunostained with antibodies against the N-terminal portion of the Syt1 C2A domain (magenta images), synaptophysin-1 (green images), and EGFP (grey images). The merged images show the presence of WT and delta HB Syt1 in the presynaptic termini identified by synaptophysin-1. Scale bars: 10 mm.(1.84 MB PDF)Click here for additional data file.


E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation.DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms N-Terminal Domain), exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271). We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions.In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays. DNA replication and cell division are probably the most fundamental processes in the cell life cycle. Both proceed through a remarkably conserved general mechanism and are inextricably intertwined to each others and to the cell metabolism .oriC, inducing DNA strand melting [E. coli, DnaA also interacts with the ring helicase DnaB and directs the loading of DnaB/DnaC onto the single stranded DNA (ssDNA) region. After binding of DnaB on the ssDNA region of the oriC, DnaC is released in an ATPase dependent manner. Then, DnaB recruits the DnaG primase and DNA polymerase III to form the replication fork [B. subtilis, two additional essential proteins, called DnaB (be aware of the confusing nomenclature between the E. coli and B. subtilis) and DnaD, are engaged in entry of the ring helicase at oriC. DnaB could function as a membrane anchoring factor for the replication initiation machinery [E. coli DnaC [oriC travel in opposite directions to achieve replication of the entire chromosome. When the replication forks reach the terminus, terC, the replication complexes are dismantled in a process involving specific termination factors [The DNA replication cycle can be divided into three distinct stages; initiation, elongation, and termination. Replication is initiated by the highly conserved AAA+ superfamily ATPase-member DnaA that binds the  melting -4. In E.achinery  or, togeoli DnaC , in the oli DnaC . DnaD inoli DnaC ,9. It exoli DnaC . During  factors .E. coli and in B. subtilis [E. coli and in B. subtilis, the molecular basis of cell division has not been as well studied in cyanobacteria. Nevertheless, studies with the two unicellular cyanobacteria Synechocystis PCC6803 and Synechococcus PCC7942 and the filamentous Anabaena PCC7120, have allowed the identification and the characterisation of clear Fts and Min orthologs as well as ZipN/Ftn2 and Ftn6, two cell division factors restricted to cyanobacteria [ftn6 deletion leads to cell division defects, resulting in cells dramatically elongated in Synechococcus PCC7942 or enlarged in Anabaena PCC7120 [ftn6 do not exhibit condensed Z-rings, but rather diffuse localization of FtsZ [The earliest event in bacterial cytokinesis is the definition of the future cell division site. This occurs through the dynamic assembly/disassembly of the Z-ring structure resulting from the self-polymerization of the ubiquitous tubulin-like protein FtsZ ,18. Placsubtilis ,18. Oncebacteria -21. Alth PCC7120 ,21, the Based on sequence and structure analyses, we here propose that the cyanobacterial-specific cell division factor Ftn6 contains a not hitherto described N-terminal domain related to the DnaD-like domain found in the DnaD chromosomal replication protein family. Identification of the Ftn6 N-terminal Domain, we termed FND , opens up very interesting perspectives about Ftn6 function in cell division and possibly in chromosome segregation as well as on their necessary interplay. Consistently, we also showed that Ftn6-depleted cells are sensitive to DNA damages generated by UV rays.Synechocystis PCC6803 Ftn6 protein sequence (Syn6803) as query. BLAST search of the NCBI-database allowed the identification of 27 Ftn6 orthologs, all belonging to the cyanobacteria phylum. No ortholog was found in plastids or other prokaryotes. Interestingly, Ftn6 orthologs were found in all Nostocales (5 out of 5), Oscillatoriales (4/4) and Gloeobacterales (1/1), in some Chroococcales (17/29), but not in Prochlorales (0/13) even in those whose genome is fully sequenced. This finding, along with the viability of ftn6-depleted mutants [18 to L94 in Syn6803 Ftn6), termed here FND for Ftn6 N-terminal Domain, is bipartite with the first 28 amino-acids (L18 to L45) poorly conserved and the 49 remaining ones (W46 to L94) characterized by the W-X3-A-X2-E-X4-G-R-Y-X3-S-X4-L-X2-W consensus . DnaD consists of two domains with distinct biochemical properties [Interrogation of protein domain databases did not allow identification of any FND-related domain (data not shown). Conversely, PSI-BLAST searches of the NCBI non-redundant database using the Syn6803 FND sequence as query identified significant hits for the alignment of this sequence with its orthologs and, interestingly, with members of the DnaD protein family. DnaD sequences that were reported from the 3th PSI-BLAST iteration share low, typically less than 20% identity, but significant similarities with Ftn6 orthologs  and from Enterococcus faecalis (PDB: 2I5U). DnaD-like domains reported from the PHYRE search share low but noticeable similarities with all FND tested (data not shown), suggesting structural similarities between FND and the DnaD-like domain. Then, Streptococcus mutans DnaD-like domain has been included in the alignment shown in Figure 50 and A54 in Syn6803 FND) and the leucine and the tyrosine (L69 and W72 respectively) at the extreme C-terminus of the fourth helix  the DNA target(s) of Ftn6 remain to be determined.Databases search of Ftn6 and DnaD domain-containing proteins were performed using BLAST e < 10-4) ,34 and P0-4 ,34 aSynechocystis PCC6803 and its derivative ftn6Δ::Kmr/FTN6+ [-2 UV radiation and incubated 7 days at 30°C under the above described light conditions.WT mr/FTN6+  were gromr/FTN6+ . Cells wMM and CS collected the data, participated in the sequences alignments and the modelling of the 3D-structures. FC and CS conceived the study, carried out the analysis of the data and drafted the manuscript. FC coordinated the study. All authors read and approved the final manuscript.Description of the Ftn6 sequences identified by BLAST. The table reports the organisms, the Genbank accession numbers and the length of the Ftn6 sequences identified by BLAST and shown in the Figures Click here for fileLOGO profile of the DnaD-like domains. The LOGO profile was generated from the ClustalW [. The red dots at the bottom of the alignment represent the hydrophobic positions conserved in the DnaD-like domain family. The 3D-structure shown at the top of LOGO profile corresponds to the DnaD-like domain of the replication proteins from Streptococcus mutans (PDB: 2ZC2).ClustalW  alignmenClustalW . The redClick here for fileSurface amino-acid conservation of the FND domain. The surface amino-acid conservation of the FND domain was calculated with Consurf [ Consurf  using thClick here for fileDescription of the DnaD sequences shown in the Figure Click here for file


Proteins, especially larger ones, are often composed of individual evolutionary units, domains, which have their own function and structural fold. Predicting domains is an important intermediate step in protein analyses, including the prediction of protein structures.We describe novel systems for the prediction of protein domain boundaries powered by Recursive Neural Networks. The systems rely on a combination of primary sequence and evolutionary information, predictions of structural features such as secondary structure, solvent accessibility and residue contact maps, and structural templates, both annotated for domains (from the SCOP dataset) and unannotated (from the PDB). We gauge the contribution of contact maps, and PDB and SCOP templates independently and for different ranges of template quality. We find that accurately predicted contact maps are informative for the prediction of domain boundaries, while the same is not true for contact maps predicted ab initio. We also find that gap information from PDB templates is informative, but, not surprisingly, less than SCOP annotations. We test both systems trained on templates of all qualities, and systems trained only on templates of marginal similarity to the query (less than 25% sequence identity). While the first batch of systems produces near perfect predictions in the presence of fair to good templates, the second batch outperforms or match ab initio predictors down to essentially any level of template quality.We test all systems in 5-fold cross-validation on a large non-redundant set of multi-domain and single domain proteins. The final predictors are state-of-the-art, with a template-less prediction boundary recall of 50.8% (precision 38.7%) within ± 20 residues and a single domain recall of 80.3% (precision 78.1%). The SCOP-based predictors achieve a boundary recall of 74% (precision 77.1%) again within ± 20 residues, and classify single domain proteins as such in over 85% of cases, when we allow a mix of bad and good quality templates. If we only allow marginal templates (max 25% sequence identity to the query) the scores remain high, with boundary recall and precision of 59% and 66.3%, and 80% of all single domain proteins predicted correctly. and we plan on running them on a multi-genomic scale and make the results public in the near future.The systems presented here may prove useful in large-scale annotation of protein domains in proteins of unknown structure. The methods are available as public web servers at the address:  Proteins, especially larger ones, are often composed of individual evolutionary units, domains, which have their own function and structural fold. Predicting domains is an important intermediate step in protein analyses, including the prediction of protein structures. In this case the prediction can be applied to each protein domain separately, decreasing prediction times, and increasing prediction accuracy especially in the absence of homologues/templates and when interactions among residues are long ranging. Although domain-domain interactions would have to be ignored when predicting domain structures separately, stages for domain-domain interaction prediction can be designed ,2 to tieExperimental structural determination methods become hard to apply when considering large proteins of many domains. In X-Ray crystallography and NMR spectroscopy difficulties often arise when protein domains are joined by less flexible boundary regions. Also, NMR structural determination errors tend to arise when the protein is very long. As a result, experimental methods often determine structures by only examining individual domains or at most a few domains together ,5.n entirely follows domain n - 1 in the sequence). The main objective of these approaches is to identify domain boundary regions. Other methods try to assign residues to particular domains when the domains are discontinuous or split across the sequence (e.g. domain n is surrounded by domain n - 1 in the sequence). Often these latter methods rely on the availability of accurate 3D models (e.g. modelled by homology), from which the structure is parsed to domains using a 3D to domain parsing algorithm. DOMpro  achieves a domain boundary recall of 49% again with ± 20 residues and correctly predicts 82.3% of single domains. DOMAC  and tempOur best template-based system (SCOP95) has boundary recall and precision of 74.0% and 77.1% (F = 75.5%) at ± 20 residues and classifies correctly 85.3% of single domain proteins. Even when we only use marginal templates  we achieve boundary recall and precision of 59% and 66.3% (F = 62.4%) and predict 80% of single domain proteins correctly. Although on different sets, all measures are roughly as good as the state-of-the-art systems DomSSEA and DOMAC.We have developed a fast system for the prediction of SCOP defined domain boundaries that takes advantage of template-based structural predictions and SCOP templates. Within the limits of comparing systems on different datasets, we have shown that our ab initio system compares well with state-of-the-art ab initio predictors. Our best template-based systems outperform the ab initio system even when poor templates are available, suggesting that not only can they be used for effective domain annotation in the presence of SCOP templates, but they may achieve state-of-the-art performances when only twilight or no templates % sequence identity to the query) are available. We have also shown that our machine learning systems outperform baselines where boundary definitions are extracted directly from the best SCOP template, or from weighed and unweighed profiles of templates. Moreover we have shown that, when high-quality contact maps are factored into the prediction via a sophisticated machine learning model it may be possible to achieve even better results. The systems are entirely automated and can be run on a genome scale on a small cluster of PCs..Our future work will focus on a number of directions: training and testing our systems on marginal templates, for instance obtained by subtler homology detection algorithms than PSI-BLAST; building a large-scale database of domain predictions to make publicly available, and to feed into the prediction loop alongside SCOP definitions; studying different domain definitions, as for instance those in CATH and PrISM; testing the hypothesis that exon information can lead to improved ab initio predictions ,53. FinaIW designed and developed all the predictors, and wrote most of the first draft of the manuscript. AJMM contributed to the design of the homology component. CM produced the final homology detection component and contributed to the manuscript. ER designed an alternative pipeline and provided constant challenges to the development of the final systems. AV assisted many phases of the development and provided useful suggestions. GP sparked the process, supervised all phases, wrote parts of the initial draft, produced the final version of the manuscript, and set up the web server. All authors read and approved the final manuscript.


Dictyostelium) or in combination with a von Willebrand factor type A (VWA) domain. For the latter protein type, which was found in a few metazoans and several distantly related protists, existence in the common ancestor of opisthokonts, Amoebozoa and excavates followed by at least eight independent losses may be inferred. Our findings thus bring further evidence for the importance of parallel reduction of ancestral complexity in the eukaryotic evolution.A recent systematic survey 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. Here we show that a different circularly permuted GTPase domain actually does exist, conforming to the pattern G3-G4-G5-G1-G2. The domain, dubbed cpRAS, is a variant of RAS family GTPases and occurs in two types of larger proteins, either inserted into a region homologous to a bacterial group of proteins classified as COG2373 and potentially related to the alpha-2-macroglobulin family (so far a single protein in This article was reviewed by Lakshminarayan Iyer and Fyodor Kondrashov. For the full reviews, please go to the Reviewers' comments section. Circular permutations have been found in a wide range of proteins [One of the evolutionary innovations that may affect a protein fold is the so-called circular permutation ,2. This proteins  and otheproteins ,4. The sP-loop GTPases and related proteins form a vast superclass of globular α/β proteins with the most conserved and functionally important regions denoted as G1 to G5 motifs . One of A recent systematic search for circularly permuted GTPases has identified the YRG family as the only group with such a modification of the GTPase domain . The autDictyostelium discoideum, EAL68747.1 and EAL60755.1 , contain a putative circularly permuted GTPase domain with the conserved motifs G1-G2 lying downstream of the G3-G4-G5 motifs , the choanoflagellate Monosiga brevicollis, the unicellular opisthokont Capsaspora owczarzaki, the amoebozoan Acanthamoeba castellanii, and the heterolobosean Naegleria gruberi  type A (VWA)" domain in the SMART collection [Acanthamoeba) upstream the VWA domain, although it receives only high E-values or is not recognised at all by domain identification tools. The CPRas protein from Naegleria additionally harbours a RING finger motif fused to the C-terminus. Interestingly, proteins remarkably similar to the C-terminal half of this CPRas type including the potential zinc finger but lacking even vestiges of an N-terminal cpRAS domain can be found in several taxa, namely Dictyostelium, Entamoeba dispar, some metazoans , and ciliates  shares with other cpRAS proteins only the circularly permuted GTPase domain corresponding to residues 2844–3013, while the rest differs completely  representing the family COG2373 in the CDD database [Dictyostelium species, D. purpureum , confirDictyostelium (EAL60755.1). We therefore suggest that the type of CPRas proteins represented by DdiCPRas2, specifically its C-terminal part, evolved by insertion of a copy of a cpRAS domain from a cpRAS-VWA protein into a COG2373-like protein introduced into the Dictyostelium lineage by horizontal gene transfer (HGT) from prokaryotes.According to our phylogenetic analysis based on an extensive sampling of the RAS family (168 sequences), all cpRAS domains are of a monophyletic origin with the cpRAS domain of DdiCPRas2 nested within the cpRAS clade, although without support from bootstrap analysis (data not shown). Given also its much wider phylogenetic distribution, it seems that the cpRAS-VWA domain organisation evolutionarily precedes the DdiCPRas2-like domain arrangement. Interestingly, when the DdiCPRas2 sequence excluding the cpRAS domain is used as a BLASTP query against the NCBI nr database, the significant hits comprise sequences from diverse bacteria but only one eukaryotic entry – another COG2373-like protein (but lacking a GTPase domain) from The YRG family is widespread in all Eubacteria, Archaebacteria, and Eukaryota ,7,11, soThere appear to be no specific or well-defined functions defined for the domains that physically combine with the cpRAS domain , so the domain architecture of CPRas proteins provides little clues as to their possible cellular roles. Their highly scattered phylogenetic distribution in a disparate set of organisms indicates that they may be implicated in rather specialised processes. The CPRas proteins are thus definitely interesting candidates for experimental characterisation.In summary, our findings correct the claim by Anand and colleagues  that theThe authors declare that they have no competing interests.ME designed the study, collected and analysed the data and drafted the manuscript, MN performed the homology modelling and contributed to discussions and writing. Both authors read and approved the manuscriptThis 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.Major comments-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 , 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. 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 .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 . 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 [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.'s group ,4define -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 . 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 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.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.Supplementary Data. A Microsoft Word document containing Supplementary Methods, Figure S1, Table S1, and supplementary sequence data.Click here for file


FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains  in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdInsP2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state. ► Talin FERM domain has a novel open conformation ► Membrane association sites are distributed along one surface of the FERM domain ► These are critical for integrin activation and focal adhesion assembly ► Simultaneous integrin and membrane binding is favored by the linear talin structure Proteins containing FERM domains are essential for a wide range of biological processes, including cell adhesion, motility, proliferation, and differentiation . FERM doThe FERM domain protein talin (∼270 kDa) plays a key role in activation of the integrin family of cell adhesion receptors and also provides a direct link between the cytoplasmic tail of the β-integrin subunit and the actin cytoskeleton. Talin consists of a ∼50 kDa N-terminal head and an elongated 220 kDa rod composed of amphipathic helical bundles . The talIn addition to the integrin binding site in F3, other regions of the talin head that are not directly involved in integrin binding are also important in integrin activation . Thus, bThe unique features of the talin head, such as the additional F0 domain which forms an intimate contact with F1 , and thefree of 25.7% were obtained after the use of translational libration screw (TLS) translation restraints during the later stages of refinement. Overall, the model contains 358 residues and 561 solvent molecules with an average B factor of 18 Å2  and 25 Å2, respectively. One molecule was observed within the asymmetric unit and is shown in Our early attempts to crystallize the talin FERM domain F1F2F3 (residues 86–400) were unsuccessful, probably because the F1 domain contains a large unstructured loop of approximately 30 residues that does not interact with other regions of the FERM domain . Recentl2, includes hydrogen-bond interactions between the guanidinium group of R181 (F1) and the carbonyl of P56 (F0), as well as the carbonyl of D104 (F1) and the indole nitrogen of W61 (F0)  and the carboxylate of E269 (F2), hydrogen bonds between the amide of V310 (F3) and carbonyl of K306 (F2), and a number of hydrophobic contacts  fold. The previously determined structures of the F0F1 (2KMA)  and F2F3W61 (F0) C. These contacts E. In con of R194 D.2 = 1.6 and 3.8, respectively), and no systematic deviations from the experimental scattering profile . Calculation of the theoretical scattering profile from the crystal structure showed a significant deviation from the experimental curve A, indica profile A. The GA profile B. Recons profile  using F0 profile B, althou profile A. The fi profile . All the1H,15N]-HSQC spectra of TH′ and the full talin head (residues 1–405) in SAXS analysis demonstrates similarity between the structure in solution and in the crystal for TH′ lacking the F1 insert loop. This loop is unstructured in the F0F1 fragment, and its removal does not induce any structural changes in that fragment . As the 1GC7), the overall domain arrangement in the two proteins is quite different. Superposition of the two structures on the F2 domain (2) while these domains are more loosely packed in TH′ (buried surface area of 620 Å2). Inspection of the TH′ crystal packing demonstrates that no symmetry-related molecules exhibit a canonical FERM cloverleaf domain arrangement through a domain-swap mechanism . Although individual domains of talin superimpose well with the equivalent regions of radixin  adopts a distinctly different β-hairpin-like conformation in the F198-S205 region B. The haThe conformation of the F1-F2 linker in TH′ is well defined, as evidenced by the electron density A, and noThe contact between the F1 and F3 domains in radixin is mediated through the positively charged side chains of R273, R275, and R279 in the N-terminal part of the helix 3α1 (F3) that form a range of ionic and hydrogen bond interactions with residues in the 1β3-1β4 loop (F1), as illustrated in Note that the above comparison only demonstrates that no stabilizing contacts between talin F1 and F3 can be formed in the relative domain orientations characteristic to ERM proteins or FAK, and does not exclude the possibility of a favorable interaction with significantly different relative orientation of F1 and F3. In addition, a “closed” form of the TH may be stabilized by interactions with the talin rod in the autoinhibited form of the molecule, or in talin complexes with other domains or proteins. If true, this would open the intriguing possibility of functional regulation of TH through large-scale domain rearrangement. The large size of the F1-F2 linker allows for repositioning of F1. In summary, the detailed structure analysis indicates that the contacts of F1-F2 linker region and the surface properties of the F3 domain favor a noncanonical linear conformation of the talin head.2 (PIP2) led to the majority of TH′ in the pellet. In the presence of pure POPS MLVs, all TH′ was associated with the vesicles. These results are consistent with the reported interaction of the ∼50 kDa talin head fragment  , and thimembrane . The linmembrane D, and thmembrane , an intebrane  at 30°C in 2YT medium. Once the cell density had reached an OD600 of 0.6, the cells were induced overnight at 18°C with 0.2 mM IPTG. Recombinant TH′ was purified by immobilized metal-affinity chromatography (IMAC). The hexa-histidine tag was removed by the addition of TEV protease overnight followed by reverse IMAC before being further purified by cation exchange chromatography. The purified protein was judged to be 98% pure by SDS-PAGE analysis. The purified protein was concentrated to approximately 10 mg/ml prior to crystallization.Mouse talin1 head domain with the loop removed  was cloned into pET151/D (Invitrogen), which contains an N-terminal hexa-histidine tag followed by a TEV protease site. Recombinant protein was expressed in the 4)2SO4 (pH 7.4). Crystals appeared as multiple thin plates stacked on top of each other within 1 week at 4°C. Further optimization of the crystals using additives proved unsuccessful, although this was not essential as the crystals could be separated from one another. Prior to data collection, a single crystal was transferred into the reservoir solution with the addition of 30% ethylene glycol, and flash frozen in liquid nitrogen. Diffraction data were collected from a single crystal at the SLS  at beamline X06SA. In total, 90 images were collected with 1° oscillations at a fixed wavelength of 0.725 Å on a Pilatus 6M detector with a crystal to detector distance of 550 mm. The data were processed and integrated with MOSFLM  was mixed with an equal volume of reservoir solution containing 100 mM Tris, 21% PEG 4,000, 200 mM  was used. A Universal Stealth RNA siRNA was used as negative control (conRNA). Subconfluent cells were trypsinized, washed in PBS, and electroporated (6 × 106 cells/ml) using a Microporator (Invitrogen) with 100 pmol of siRNA. Cells were grown on tissue culture plastic for 72 hr, and replated at a density of 4 × 104 cells on 16 mm glass coverslips for image analysis a further 24 hr later. Where appropriate, cells were cotransfected with the full-length mouse talin1 cDNA cloned into pEGFP-N1 (Clontech) as described elsewhere (2 and 3 mM EGTA) for 10 min at room temperature and mounted on glass slides. Images were taken with a 40× oil immersion objective on an inverted Nikon TE300 microscope equipped with a Hamamatsu ORCA-ER digital camera, and an X-cite 120 fluorescence illumination system controlled by Improvision's Openlab software. Note that the ability of GFP to dimerize could potentially change the properties of GFP-tagged talin1. siRNA depletion of talin1 in human endothelial cells caused defects in cell spreading and FA assembly. These were fully reversed by expression of GFP-tagged full-length mouse talin1 indicating that the GFP-tag had no obvious affect on the function of the expressed protein.Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell at passage 2 and grown according to the manufacturer's instructions in Endothelial Cell Growth Medium 2 (PromoCell) at 37°C in 10% COlsewhere . Cells w


Domain rearrangements in the innate immune network of amphioxus suggests that domain shuffling has shaped the evolution of immune systems. Branchiostoma floridae), whose genome was recently sequenced, occupies a unique position in the evolution of innate immunity, having diverged within the chordate lineage prior to the emergence of the adaptive immune system in vertebrates.Regulation in protein networks often utilizes specialized domains that 'join' (or 'connect') the network through specific protein-protein interactions. The innate immune system, which provides a first and, in many species, the only line of defense against microbial and viral pathogens, is regulated in this way. Amphioxus  that bypass usual signal transduction pathways.Domain rearrangements can potentially alter the topology of protein-protein interaction (and regulatory) networks. The extent of such arrangements in the innate immune network of amphioxus suggests that domain shuffling, which is an important mechanism in the evolution of multidomain proteins, has also shaped the development of immune systems. Caenorhabditis elegans, Drosophila melanogaster, and human, display strong conservation of many elements of these two networks. In genome evolution, domain recombination events, such as fusion and fission, can create proteins with novel domain combinations that may lead to new functions, including providing new connections inside an existing network or between different networks -WD40s , TIR-sterile alpha motif (SAM), TIR-Laminin and so on, which potentially could mediate immune-related functions, have also been identified in the amphioxus genome.Despite the presence of unusually complex patterns of repetitive DNA, the current assembly of the amphioxus genome is generally highly reliable ; notwithThe recovery of transcripts corresponding to the 'direct connector' genes is, in itself, important as many of these genes most likely exhibit developmental stage-specific expression, may be expressed in relatively low abundance, and/or are transcribed in cells that are present in relatively low numbers or are undergoing apoptosis. Efforts to locate the expression of hybrid genes are currently underway.The large-scale expansion of several families of innate receptors in amphioxus parallels that seen in sea urchin and is a shared feature of both sides of the deuterostome split. The phenomenon of lineage-specific gene expansion has also been reported for protein families in other genomes . FurtherMany of the domain combinations in amphioxus are present in separate proteins in vertebrates that are interconnected by multistep signaling pathways . Taken Two aspects of genome architecture and complexity influence innate immunity in amphioxus. First, large-scale gene expansion, a characteristic shared with sea urchin, creates a greater level of potential specificity in several families of innate immune receptors than is found in species with adaptive immune systems and could result in refinement of immune function. Second, novel domain architectures and, in particular, direct connections (shortcuts) in regulatory pathways can introduce a more refined level of functional integration of networks than would likely be achieved by the simple duplication and subsequent divergence of genes encoding immune receptors. A model for expansion and the possibility of topology change of a network is presumed in the analyses of the amphioxus genome presented here. A corollary issue raised by these observations is whether specific features of the amphioxus genome, such as the extraordinary level of site variation and unusually complex patterns of repetitive DNA, factor in such changes. Irrespective of their origins, genes with novel architectures in amphioxus could potentially serve as a pathway-level 'Rosetta stone' for elucidating new regulatory connections in the innate systems of contemporary vertebrates, similar to approaches that are used to elucidate protein and regulatory complexes in prokaryotic genomes . AssuminBranchiostoma floridae) were obtained from the JGI [N. vectensis). The genome assembly Spur_v2.0 and the GLEAN3 gene models for the sea urchin (Strongylocentrotus purpuratus) were obtained from the Baylor College of Medicine Human Genome Sequencing Center [Xenopus, zebrafish, fugu, tetraodon, ciona, nematode (C. elegans), and fruit fly (D. melanogaster) were downloaded from Ensembl [The v.1.0 genome assembly and related gene models of amphioxus .JGI-predicted models were used to develop PCR strategies for identifying cDNA transcripts. The predicted transcripts were placed onto the current assembly (v.1.0) using local BLAST (v.2.2.11) to verify genomic organization . Primers were designed  to spanASC, apoptosis-associated speck-like protein; CADO, Comparative Analysis of Protein Domain Organization; CARD, caspase recruitment domain; CID, chemically inducible dimerization; DD, death domain; DED, death effector domain; JGI, Joint Genome Institute; LRR, leucine-rich repeat; NACHT, domain present in NAIP, CIITA, HET-E, and TP1; NAIP, neuronal apoptosis inhibitory protein; NB-ARC, nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4; NLR, NOD-like receptor; PAAD, pyrin, AIM (absent-in-melanoma), ASC, and DD-like; PYRIN, amino-terminal domain of protein pyrin; SAM, sterile alpha motif; SARM, sterile α and HEAT-Armadillo motifs containing protein; SRCR, scavenger receptor cysteine-rich; TIR, Toll/interleukin-1 receptor; TLR, Toll-like receptor; TNFR, tumor necrosis factor receptor; WD40, Trp-Asp 40.QZ performed the sequence and domain analyses and prepared the figures. CMZ performed phylogenetic analyses. LJD developed approaches for identifying hybrid transcripts. MGM cloned and sequenced hybrid transcripts. YY contributed to the domain analyses of the predicted proteins. GWL interpreted immunology concepts. AG formulated the problem and planned the work. All authors contributed to the interpretation of the results and to writing of the paper.The following additional data files are available. Additional data file TIR domain containing sequences in different genomes.Click here for fileNACHT domain containing sequences in different genomes.Click here for fileSRCR domain combinations in different genomes.Click here for filePartner domains that combine with individual TIR or NACHT domains in different genomes.Click here for fileSelected JGI-predicted amphioxus gene models that have been verified by RT-PCR.Click here for fileExamples of novel domain combinations in amphioxus that represent the shortcuts between two or more proteins present in human.Click here for fileAlignment of sequences in the vicinity of the catalytic center of the caspase domain from human caspases and amphioxus proteins with TNFR-caspase or LRRs-caspase architectures.Click here for file