The β-grasp fold (β-GF), prototyped by ubiquitin (UB), has been recruited for a strikingly diverse range of biochemical functions. These functions include providing a scaffold for different enzymatic active sites (e.g. NUDIX phosphohydrolases) and iron-sulfur clusters, RNA-soluble-ligand and co-factor-binding, sulfur transfer, adaptor functions in signaling, assembly of macromolecular complexes and post-translational protein modification. To understand the basis for the functional versatility of this small fold we undertook a comprehensive sequence-structure analysis of the fold and developed a natural classification for its members.As a result we were able to define the core distinguishing features of the fold and numerous elaborations, including several previously unrecognized variants. Systematic analysis of all known interactions of the fold showed that its manifold functional abilities arise primarily from the prominent β-sheet, which provides an exposed surface for diverse interactions or additionally, by forming open barrel-like structures. We show that in the β-GF both enzymatic activities and the binding of diverse co-factors (e.g. molybdopterin) have independently evolved on at least three occasions each, and iron-sulfur-cluster-binding on at least two independent occasions. Our analysis identified multiple previously unknown large monophyletic assemblages within the β-GF, including one which unifies versions found in the fasciclin-1 superfamily, the ribosomal protein L25, the phosphoribosyl AMP cyclohydrolase (HisI) and glutamine synthetase. We also uncovered several new groups of β-GF domains including a domain found in bacterial flagellar and fimbrial assembly components, and 5 new UB-like domains in the eukaryotes.Evolutionary reconstruction indicates that the β-GF had differentiated into at least 7 distinct lineages by the time of the last universal common ancestor of all extant organisms, encompassing much of the structural diversity observed in extant versions of the fold. The earliest β-GF members were probably involved in RNA metabolism and subsequently radiated into various functional niches. Most of the structural diversification occurred in the prokaryotes, whereas the eukaryotic phase was mainly marked by a specific expansion of the ubiquitin-like β-GF members. The eukaryotic UB superfamily diversified into at least 67 distinct families, of which at least 19–20 families were already present in the eukaryotic common ancestor, including several protein and one lipid conjugated forms. Another key aspect of the eukaryotic phase of evolution of the β-GF was the dramatic increase in domain architectural complexity of proteins related to the expansion of UB-like domains in numerous adaptor roles.This article was reviewed by Igor Zhulin, Arcady Mushegian and Frank Eisenhaber. The discovery of covalent modification of eukaryotic proteins by the conjugation of ubiquitin to the ε-amino groups of target lysines has spawned some of the most exciting directions of research in current molecular biology -3. UbiquThe centrality of Ub conjugation in eukaryotic molecular biology has led to numerous investigations on Ub and Ub-related domains ,10. Thes12 and other solutes in animals and bacteria  the TGS domain, an RNA-binding domain found in aminoacyl tRNA synthetases and other translation regulators . 2) C, 2FUGS -36). 4) C, 2FUGS ). 5) FunC, 2FUGS ) and RnfC, 2FUGS . 6) ConsC, 2FUGS  and bactC, 2FUGS -43). 7) C, 2FUGS ). 8) MutC, 2FUGS . These obona fide conjugation systems that transfer β-GF proteins to target polypeptides [One notable evolutionary question in this regard was the origin of eukaryotic Ub and its relationships to other domains with the β-GF. The first major advances in this direction came with the identification of the sulfur transfer proteins, ThiS and MoaD, respectively involved in thiamine and Molybdenum cofactor (MoCo) biosynthesis, which contained β-GFs closely related to Ub ,47. Furtpeptides . Hence, With some clarity emerging on issue of the origin of Ub/Ubls and the associated biochemical networks, we sought to investigate the broader issue of the adaptive radiations of the entire β-GF. In particular we were interested in a number of problems from structural and evolutionary stand points: 1) Establishing the entire gamut of structural and topological variations that have emerged in the β-GF. 2) Identifying any unifying structural themes that might exist across most or all functionally diverse versions of the fold. 3) Determination of the lineage-specific sequence-structure correlates for the varied functional adaptations of the β-GF. 4) Developing a higher order evolutionary classification for the β-GF and using it as a scaffold to identify the major temporal phases of adaptive radiation. 5) Identifying instances of drastic shifts in biological or biochemical functions in specific monophyletic lineages of the β-GF. One example of such a functional shift is seen in the evolution of the classical Ub-like proteins, where a unique post-translational modification system emerged from a core metabolic sulfur transfer system. 6) Identifying previously unrecognized members of the fold, if any, and thereby expanding the functional spectrum or providing a rationale for function prediction of uncharacterized members of the fold. 7) We also hoped that the β-GF might provide a model for understanding the more general problem of how certain small protein folds tend to be extensively deployed in a whole diversity of functional contexts.In this article we present the results of our systematic analysis of the β-GF with the objective of addressing the above points.As the β-GF is small in size and its representatives very divergent, it is not possible to exhaustively identify all members through sequence or structure similarity searches initiated from a single starting point. Accordingly, we used a multi-pronged strategy of sequence, structure, and topological similarity searches. As a starting point, we used all the currently available structures of β-GF proteins from the Protein Data Bank (PDB) . This sebona fide β-GF domains were compared in order to establish a unique core template topology that discriminated the β-GF from all other folds . This vAll other versions of the β-GF are characterized by major modifications to the 4-stranded core in the form of distinct inserts that add new secondary structure elements. The first of these is a previously uncharacterized variation containing an insertion of one or more strands between the helical segment and strand 3. The conserved inserted strand seen in all domains with this version forms a hairpin with the connector segment between the helical segment and strand 3 which also assumes an extended conformation. This hairpin, together with any additional strands in the insert results in these versions of the fold assuming barrel-like structures with differing degrees of openness Figure , Table 1The most common version of the β-GF is typified by the presence of an additional strand that packs against the conserved third strand at the margin of the core β-sheet. The acquisition of this additional strand has resulted in the emergence of a connector arm that joins it to the terminal conserved strand of the core sheet Figure , Table 1The 5-stranded versions may show further variations due to inserts at different points in the conserved core. One prominent example is the 2Fe-2S ferredoxin, which contains an insert before the third conserved strand with conserved cysteines for chelating the Fe ion. Similarly, a long insert adopting an extended conformation is observed at a comparable position in several versions of the SLBB domain  and the Our structure similarity searches identified a few structures which, despite lacking the core conserved topology of the classical β-GF, aligned well with a part thereof. Reciprocal searches indicated that β-GF domains were the best hits for these structures. Additionally, these structures were not representatives of any other previously identified folds. These structures include the S4 RNA-binding domain (PDB: 1c05 ), the WWIn order to address the prime evolutionary questions about the β-GF, we attempted to construct a classification that most closely approximates the higher-order evolutionary relationships of the members of this fold. The small size of the majority of the versions of this domain often precludes sufficient resolution of relationships using conventional phylogenetic tree methods, sometimes even within superfamilies that display significant sequence similarity. This difficulty is further compounded by the extreme sequence divergence even between versions having highly similar tertiary structures (e.g. ubiquitin and ThiS). Hence, we had to rely to a greater extent on structure similarity-based clustering, shared derived structural characters, and phyletic patterns of sequence superfamilies to reconstruct the evolutionary history. Thus, we produced the classification using the following general steps: 1) sequence similarity-based clustering with the BLASTCLUST program  helped iThe above analysis of the structural diversity of the fold suggests that the 4-stranded version is the simplest form from which all other versions could have been derived through accretion of inserts and additional secondary structure elements. Two structurally close superfamilies of the 4-stranded β-GF domain, namely the IF3-N and the archaeo-eukaryotic RNA polymerase domain, are respectively universally conserved in the bacterial and archaeal-eukaryotic branches of life. This, taken together with their shared general functional connection to RNA metabolism, suggests that they arose from a similarly structured precursor that can be traced back to the last universal common ancestor (LUCA). This structurally simple representative of the β-GF is likely to represent one of the most basal lineages of the fold. The remaining sequence clusters BofC, yeast Yml108w, and immunoglobulin-binding proteins of low GC Gram-positive bacteria with structurally comparable, simple 4-stranded β-GF domains show extremely limited phyletic patterns , the riThe FimD superfamily, while containing a unique structural variant of the fold, shows greatest structural similarity to the above assemblage. Its phyletic pattern is limited, being found only in proteobacteria and deinococci . These families include six conjugated versions  ,80 and sIn bacteria, Ub-like superfamily includes several sporadically distributed UB-like families which have been previously described in considerable detail . SeveralThe second major clade of the 5-stranded assemblage unifies a group of β-GF domains whose interrelationships were previously unknown. This clade is unified by the presence of a set of inserts that are associated with binding soluble ligands or chelating metal ions. While the inserts themselves are poorly conserved in sequence, their position, especially in relation to the bound ion or ligand, is well conserved. The main sequence superfamilies in this clade are the 2Fe-2S ferredoxins, the SLBB domains, and the molybdopterin-dependent oxidoreductase domains. As recently shown, the SLBB superfamily is of bacterial provenance . The molA distinctive superfamily of the 5-stranded assemblage that we discovered in our analysis was the N-terminal module of the aldehyde oxidoreductase (AOR-N) (PDB: 1AOR ) that coThe inference of at least 7 β-GF or β-GF-derived the S4 domain) lineages in LUCA suggests that there was a major diversification of the fold even before LUCA Figure . In stru domain lA corollary to the inference of the ancestral function of the fold is that there were major functional innovations even in the pre-LUCA period. These are most prominently seen in the 5-stranded assemblage, and appear to be associated with the emergence of distinctive roles in sulfur delivery and scaffolding of Fe-S clusters. Previous observations have shown biochemical links between the formation of metal-sulfur clusters and sulfur transfer, including pathways in which ThiS and MoaD-like proteins participate . This obThe emergence of the two prokaryotic superkingdoms, the archaea and bacteria, was marked by numerous superkingdom-specific innovations. Several of these innovations appear to have happened early in the history of the bacteria followed by multiple lateral transfers to the archaea. Likewise, innovations occurring in bacteria were also transferred to eukaryotes both during the primary endosymbiotic event and sporadically through later transfers. Members performing some form of most of the biochemical functions observed in extant representatives of the fold emerged in course of the post-LUCA diversification in bacteria. In certain cases there were no major shifts in basic biochemical activity but only an expansion of the range of specific biological contexts in which these activities were deployed. These included new RNA-binding/ribonucleoprotein-related contexts emerging within diverse branches of the clade (e.g. L25 in the fasciclin-like assemblage and the prokaryotic UB-like family fused to Mut-7C RNAse) or adaptation of ThiS/MoaD-type proteins in sulfur transfer systems related to synthesis of lineage-specific metabolites . The priThis phase also saw the recruitment of several forms of the β-GF domain for mediating specific protein-protein interactions in the assembly or stabilization of multi-protein complexes. Different distantly related β-GF domains were recruited in the biogenetic systems of flagella and analogous structures, the pili. The FimD protein has an N-terminal β-GF domain fused to a C-terminal outer membrane-spanning domain . This β-However, the most important innovation in the bacteria was the emergence of potential conjugation systems that covalently linked ubiquitin-like β-GF domains to other proteins (predecessors of the eukaryotic conjugation systems). In functional terms, this process represents a collusion of the sulfur-transfer aspect with the protein interaction function which was also widely emerging in members of the fold. The preliminary analysis of these bacterial UB-like systems suggests that they might have already acquired roles related to protein stability and signaling. The details of the bacterial antecedents of the eukaryotic UB-conjugation system have already been discussed in a recent work  and are Genomic and cell biological evidence suggests that the eukaryotes emerged as a result of a basic endosymbiotic event between a proteobacterium and an archaeon (most likely a euryarchaeon) -97. ConsSubsequently in eukaryotic evolution, there appear to have been several innovations of non-conjugated versions. Many of these continued to function in contexts related to UB signaling, presumably by recognizing conjugated UB moieties Figure . HoweverPrevious studies on domains occurring in diverse architectural contexts in multi-domain proteins have hinted at a strong relationship between domain architectures and functional constraints . We systIn contrast, versions involved in protein and nucleic acid interactions are under no major constraints to remain as standalone forms of the domain. Hence, we find numerous instances of β-GF domains involved in this function occurring in multi-domain architectures. The ribosomal proteins tend to be small and usually one or two-domain proteins. Accordingly, there is not much architectural diversity seen in case of forms like L25. The forms found in the DNA-dependent RNA polymerase represent some of the most complex architectures wherein the β-grasp domain is inserted within an RRM-fold domain which in turn is inserted within a larger, multi-domain scaffold . In mostMycoplasma and an inexplicably high architectural complexity in Thermotoga maritima. Thus, barring very few exceptions, the main tendency in prokaryotes is a wide variability in the number of proteins with β-GF domains rather than any concerted increase in architectural complexity.To objectively assess the trends in domain architectural complexity, we made use of the previously devised complexity quotient (CQ) . The CQ Trichomonas vaginalis, a basal eukaryote, which appears to have undergone a massive, relatively recent proliferation across most protein families [Eukaryotes not only have greater numbers of β-GF domain proteins, but also appear to display greater diversity of domain architectures relative to the prokaryotes. The complexity of the β-GF proteins as well as their numbers appear to increase throughout eukaryotic evolution with the highest figures observed in multicellular organisms of the eukaryotic crown group. However, the increase in architectural complexity is not linear across eukaryotes, with a tendency to plateau in animals. The only exception to the strong trend is families . As a refamilies .Typically, many of the eukaryotic multi-domain architectures, both ancient and lineage-specific, tend to combine the UBL domains with other signaling domains, typically those involved in UB-signaling. These combinations include those with deubiquitinating peptidases (e.g. of the OTU superfamily), E3 ligases usually of the RING superfamily Figure , and othWe next sought to decipher the relationship between functional diversification and structural elaborations of the fold. For this purpose, we created an idealized representation of the β-GF fold Figure  and diviLittle is known of the exact mode of interactions of the basal 4-stranded versions of the fold. However, the apparent rarity of the simple 4-stranded versions suggests that there appears to be a tendency to elaborate the core sheet to provide an increased interface for interactions. On the whole, the exposed face mediates more interactions across the β-GF fold compared to the obscured face. Thus, the proliferation and widespread utilization of the 5-stranded version might be associated with the availability of a larger surface on the exposed face for mediating contacts. Another evolutionary trend is the formation of a barrel-like configuration through insertion of strands which on instances provides a classical interaction interface at the open end of the barrel. We discuss below more specific themes of interaction that were observed in multiple superfamilies of the fold.As discussed above, the prevalence of soluble ligands such as sugars, amino acids, and metabolic intermediates for different sequence superfamilies of the clade suggested an ancestral solute-binding role for these proteins. Analysis of the interactions with respect to the shared structural core of this assemblage suggests that the insert and the lateral shelf form an interface for soluble ligand interaction in fasciclin, GS-N, and phosphoribosyl-AMP cyclohydrolase domains ,77,109. 12-binding proteins contain a conserved aromatic residue in the pre-strand 3 insert which plays a central role in binding the ligand [The unifying inserts of this clade typically occur in the region prior to strand 3 and in the region associated with the connector arm or the additional strand of the 5-stranded core. However, there is considerable diversity in the means by which these inserts mediate specific interactions, both between and within different superfamilies of this clade. The 2Fe-2S ferredoxins contain a characteristic set of four cysteines, three of which come from the pre-strand 3 insert and one from the connector arm-associated insert which help in coordination of the 2Fe-2S cluster . The proe ligand . In the e ligand . The aboe ligand ,115.The AOR-N domain represents the second independent case of a β-GF domain acquiring the capability to bind tungstoperin or molybdopterin and iron-sulfur clusters (4Fe-4S). Here, a head-to-tail dimer formed by the two tandemly repeated β-GF domains bind metallopterin via the unusually structured connector arms that form a strand at the fringe of the core 5-stranded sheet  Figure . While tA diverse range of protein-protein interactions are shown by both prokaryotic and eukaryotic members of the 5-stranded assemblage, including those with the E1, E2 and E3 enzymes or their prokaryotic counterparts. The recently published structure of the complex of Nedd8 with its E1 and E2 enzymes , in conjIn the complex of Nedd8 with its conjugating enzymes, the Nedd8 moiety covalently linked to the cysteine in the thioester-forming α-helical domain of the E1 protein also serves to recruit its specific E2 . This ocIn contrast to protein-protein interaction, little is known of the actual mode of RNA-interaction by versions of the classical 5-stranded assemblage like the TGS domain. While it is possible that the exposed face provides an interface as observed in the L25-5S rRNA-interactions , the conAt least 3 distinct superfamilies of enzymes use the β-GF the primary scaffold for their active sites. None of the enzymatic forms can be confidently traced to LUCA. Instead they appear to have emerged early in bacterial evolution. Furthermore, the enzymatic versions are only distantly related to each other suggesting that the β-GF fold has been convergently used to provide catalytic scaffolds. In the case of phosphoribosyl-AMP cyclohydrolase and the molybdopterin-dependent oxidoreductases like sulfite oxidase, the main pre-adaptation for catalysis stems from the ancestral ligand binding capabilities that emerged in the assemblages to which they belong. In the case of the Nudix superfamily, the pre-adaptation was possibly the nucleic acid-binding ability of the ancient 4-stranded versions of β-GF. Consistent with the convergent origins of catalysis in each instance emergence of enzymatic activity appears to have resulted in some distinctive structural changes that go beyond the above pre-adaptations shared with ligand-binding forms. In the molybdopterin-dependent oxidoreductases, the structural innovation is in the form of an elaboration of the ancestral ligand-binding features – the extensive inserts resulting in the 3-layered structure with an open barrel-like element provide a pocket for the cysteine-attached co-factor as well as the substrate.In the phosphoribosyl-AMP cyclohydrolases a sequence alignment and super-position of the conservation onto the available structures indicates two basic active site elements . The PXG motif in strand-2 immediately after this "outflow" also provides the space to accommodate substrates due to a lack of large side-chains. Additional substrate contacting positions also come from the rest of the exposed face. However, the conserved residues actually required for catalysis do not come from the exposed face, but from the helical segment and loop connecting strand-2 to it Figure . This loA few modes of interactions thus far appear to be restricted to certain sporadic lineages or are only seen in certain highly derived forms of the domain. The superantigen/toxin-type and strepto/staphylo-kinase-type β-GF domains share an unusual general mode of interaction with plasmin and the T-cell receptor β-chains, respectively. Both appear to contact partners "side-on" via the edge of the domain on which the additional strand of the 5-stranded versions is situated. A comparable "side-on" interaction is also seen in the evolutionarily distant 4-stranded form of the fold found in the Ig-binding domain. However, this latter interaction is distinct in having additional extensive contacts supplied by the helical segment from the obscured face of the domain Figure . It is uWhile the β-GF has been thoroughly investigated in the context of the interactions of ubiquitin and UBLs in eukaryotes and their prokaryotic relatives like ThiS and MoaD involved in sulfur transfer, the broader evolutionary history of the fold was poorly understood. We sought to redress this by developing a natural classification for the fold and using it as guide for exploring the tempo of its evolutionary radiations and details of its functional adaptations. As a result we identified several novel members of the fold, including some distinctive previously unidentified modifications. The reconstruction of the evolution of the fold suggests that the major structural variants and some of the basic biochemical features and modes of interaction had emerged prior to LUCA. This suggests that even before the radiation of the extant lineage of Life there were several rounds of duplication followed by extensive divergence, including major structural changes.The scenario emerging from our analysis also suggests that the earliest reconstructed function of the β-GF domain was in the context of ribonucleoprotein complexes, probably as an RNA-binding domain. Based on the functions of extant versions of the domain, like the TGS superfamily, the IF3-N domain, and early structural derivatives such as the S4 superfamily, it is quite possible that the earliest versions of the fold played a generic role in a primitive pre-LUCA translation system. Thus, the earliest diversification events of the β-GF fold likely occurred in the context of the RNA-world, probably with the acquisition of increasingly specialized roles in the evolving translation apparatus. Amongst the major pre-LUCA functional shifts were those relating to the biosynthesis of sulfur-containing compounds and scaffolding of Fe-S clusters. On the face, such functional shifts from earlier roles in translation-associated RNPs appear drastic and puzzling. However, it should be noted that there is a functional connection between the sulfur incorporation pathways of thiamine biosynthesis and thiouridine synthesis in RNA ,120. HenThe major post-LUCA phases of the evolutionary history of the β-GF fold saw two major spurts of innovation. The first, occurring primarily in the bacteria, was accompanied by an extensive exploration of the biochemical function and interaction space by different versions of the fold. This was marked by the acquisition of diverse soluble ligand-binding capabilities through distinctive structural modifications as well as extensive deployment in different protein-protein interaction contexts. Most notably, the scaffold on at least 3 independent occasions acquired very different enzymatic activities even though the β-GF fold did not ancestrally support catalytic activities. The eukaryotic phase did not see extensive innovation in terms of fundamentally different biochemical functions, but the diversity of protein interactions within the ubiquitin-like superfamily of the 5-stranded assemblage was vastly expanded through extensive sequence divergence of the primary interaction surfaces of the superfamily. This phase was also accompanied by ongoing innovation of new multi-domain architectures associated with the eukaryotic expansions of Ub-like signaling domains  database of protein sequences  was searched with the BLASTP program . Profilebona fide β-grasp domains. If a complete match was obtained these structures were tagged as congruent, while those which were not were ranked in descending order of elements that did not match. This discrimination of the potential candidates was further confirmed by visual examination of each structure. The interacting residues of various proteins of β-grasp fold with their interacting molecules have been deduced using custom made PERL scripts. The scripts encode interacting distance cut-off values of 5.0 Å and 3.5 Å between appropriate atoms in 3-D for deducing the hydrophobic and polar interactions respectively. These inferred interactions were further examined manually using Swiss-PDB viewer for confirming the contacts between amino-acid residues of β-grasp fold proteins and atomic groups of interacting partners.Structure similarity searches were conducted using the standalone version of the DALI program ,55 with Igor B. Zhulin, Oak Ridge National Laboratory and University of Tennessee, USABiology Direct 2007, 2:4). In this investigation, authors launched a large-scale exploration of the β-grasp fold diversity and evolution. This is an important, but daunting task, because the fold is well populated and comprises divergent domain families. In my opinion, both strengths and weaknesses of the paper are resulting from this fact.The current study is related to the previous work by the authors reporting a novel domain superfamily within the β-grasp fold , it will take some time... Ideally, all information pertinent to the known facts should be introduced in the Introduction and Discussion, whereas Results should show clearly what new information has been obtained in this study. I think combining Results and Discussion in one section creates this feeling that known and novel facts are mashed together. I know that it is much easier for authors to present such a complex study in this way, but the trade-off is the lack of above mentioned distinction. One suggestion on improving the overall organization of the current scheme is to introduce numerical headings and subheadings. For example, in the current version of the manuscript the reader is left to wonder whether "Basal versions of the β-GF" is a subheading of "Natural classification of domains" or they are equal headings, and so on. Having 1., 1.1, 1.1.1. system should help to see the logic of the story better.In my opinion, the weakness of this manuscript is in its organization. Potentially, this can be improved, although I keep the door open for a counterargument by not providing constructive suggestions on how to do that. Because there is such a large body of information, its organization is important. As for any scientific contribution, it is imperative to identify what is the status quo in the field and what has been done by the investigators to advance our knowledge. This particular paper is written as mix of a thorough literature review (there are 162 references!) and an original study. The purpose of the former is (hopefully) to synthesize novel ideas/leads from numerous known facts, whereas the purpose of the latter is to generate novel facts filling the gaps in knowledge. This paper does both, but it is does not Given the intricate connection between the results and inferences drawn from them, we felt it better to combine the Results and Discussion sections to avoid redundancy. We have now reworded the text to more clearly differentiate the discoveries presented in our work from those that are already known. Accordingly, we have also revised the heading style and added subheadings, where appropriate to improve clarity. While we could have used a numbered style for greater clarity as recommended by the referee this scheme does not fit into Biology Direct's manuscript style.Essentially half of the manuscript is devoted to developing a scenario for the fold evolution. This scenario is certainly plausible, as there are no obvious flaws or contradictions. One the other hand, the starting point of inferring relationships is structural information and so is the end point of their verification. I might be wrong here, but this is the feeling. I wish we would have an independent method (or better methods) of testing these relationships more rigorously.Given the small size of the domain, the absence of catalytic, active site residues and the level of sequence divergence several superfamilies of the β-grasp fold can only be unified by structural features. As a result, shared structural features are a principal guide for developing the evolutionary at a higher level scenario. However, as mentioned in the text, we also used distance trees based on pairwise Z-scores of structural comparisons to infer evolutionary relationships between different superfamilies. Our final classification is thus a parsimonious reconstruction that includes information obtained in both these methods. Thus, what is presented is a phylogenetic hypothesis that is amenable to future falsification. The utility of this phylogenetic hypothesis is in developing a framework for exploring emergence of various properties of different superfamilies of the fold. We do appreciate the referee's concern for the means for independent falsification of the presented hypothesis. We believe these do exist even if not straight forward: 1) Availability of new structures can allow testing of the suggested scenarios to present evidence for and against the scenario presented here. 2) The scenario presented here makes predictions of certain functional properties such as binding interfaces and substrates that explicitly stem from the phylogenetic analysis. Testing these predictions allows a test of the underlying scenario.2. On page 6 there is a reference to HMM searches initiated with models built from multiple alignments; however, there is no mentioning of the HMMER package in the Materials and Methods.We have added a brief description of HMM searches to Materials and Methods.Arcady Mushegian, Stowers Institute for Medical Research, Kansas City, USAThroughout the paper, consider changing "hits" to "matches" in the context of BLAST searches: "hit" used to have a special technical meaning, e.g. "using the two-hit method to trigger ungapped extensions".We admit this possibility of potential misunderstanding and make the suggested change.Pg 5 of the pdf, 4th ln from the bottom: what is "topological similarity search"? It is not defined in M&M.Pg 6, par 2, ln 8. What is "congruence to the unique topological template", how such congruence was evaluated, and what if something is "incongruent"?We have added a description of this procedure to the Materials and Methods. We now describe how congruence was evaluated.Pg 7 ln 5–6. Conserved pattern of hydrophobic interactions in the core is said to strongly support its monophyletic origin. I am wondering what is the null hypothesis and the burden of proof here: surely, there are also some weakly conserved and non-conserved hydrophobic interactions – are there more of them than the conserved ones? If so, do they reject the hypothesis of the monophyletic origin? Is there any test that can reject such a hypothesis for proteins with a shared fold?With respect to ancient folds  there are two main scenarios, both of which based on our current understanding are possible: 1) Monogenesis of all ancient folds, or at least major subsets of them, like the α+β two layered fold category to which the β-GF, RRM, ASCR etc belong.2) Polygenesis in which various distinct α+β two layered folds have independently arisen and convergently adopted similar general structural features. If the former were the case, then the hypothesis of domains not sharing a fold cannot be rejected in an absolute sense. However, one could still define exclusive monophyletic groups.Under both scenarios we believe that the null hypothesis would be that non-monophyly (or exclusive monophyly) of two or more distinct domains with the burden of proof being the need provide evidence for their exclusive monophyly. Under both scenarios an exclusive monophyletic group or a distinct fold would have a unique conserved network of hydrophobic interactions. This concept is an extension of a monophyletic protein superfamily defined based on sequence similarity: the superfamily has a distinctive constellation of conserved set of residues – similarly, there are distinct conserved networks of hydrophobic residues that are present in the β-GF or the RRM fold. Just as in the sequence alignment the presence of many non-conserved positions does not reject the monophyly of the aligned sequences, there being many non-conserved interactions in the hydrophobic network does not reject the monophyly of domains with a shared fold. However, the absence of the conserved network characteristic of a fold in domain casts doubt on its belonging to the fold. This is especially so, if there are notable structural differences and a hydrophobic network that corresponds to some other fold. For example the BtrG-like proteins (1vkb A) and the tRNA ribosyltransferase-isomerase 1vky B show some general similarity with the β-GF. However, they lack the conserved hydrophobic core shared by the β-GF. The former has instead emerged from an internal repeat of simpler units and the later might be interpreted as distinct barrel with different stabilizing interactions. Hence with current data these are rejected as members of the β-grasp fold.Pg 12 par 3: "The strong conservation of this unique structural feature as well as the exclusive grouping of these versions in structure similarity-based clustering": are those indeed two independent lines of evidence, or is the latter a direct consequence of the former?We did not imply anywhere that these two are entirely independent forms of evidence. We agree that the presence of a shared strand does contribute to increasing the structural similarity of the 5-stranded group. However, their clustering based structural similarity Z-scores does not exclusively depend on the presence of that additional strand alone. There are other more subtle, or "distributed" elements of similarity, which contribute to their exclusive clustering. In an experiment in which a representative was chosen from each lineage in figure 3, the strand 5 left out, and then clustered using the MUSTANG program we still recover their exclusive grouping supporting the above-stated contention.Pg 15 par 2: What is "principle of phylogenetic bracketing"? If it is the same as "Extant Phylogenetic Bracket" attributed to L. Witmer, then this is not a suitable method for inferring ancestral function, in particular when it is distributed sporadically in the extant lineages.Witmer's extant phylogenetic bracketing is used to infer a feature of an extinct clade which is bracketed by the features in two extant clades with the underlying assumption of the parsimony principle. We do not imply the above on page 15, but admit that our usage of the term would result in confusion with the above. Accordingly, we modify the discussion in this regard to avoid using the potentially confusing term "phylogenetic bracketing".Text and Fig. The reviewer is absolutely correct, we in no way intended to refer to these lineages as basal versions, but rather sporadic versions potentially derived from more widely distributed 4-stranded versions. We have altered to text and the label in Fig. 3to represent this point more accurately.Pg 31 ln 6 of M&M: it is "compositionally biased" not "computationally biased".We have made the correction.Frank Eisenhaber, Institute of Molecular Pathology, Vienna, AustriaThe work by Burroughs et al. is a careful, virtually encyclopedically complete collection of β-GF protein segments in sequence databases and a meticulous analysis of the associated structural and functional knowledge in the literature. For the reviewer, it was especially interesting to follow the discussion of the authors how the splitting of evolutionary lines was associated with critical evolutionary events  and how the functional capabilities of the fold got realized during history of life and how they are associated with elaborations of the basic fold.The value of this work for the annotation of uncharacterized sequences could be enhanced if the authors made some of their results publicly available in electronic form  as supplement to this paper.Within the provided additional file is a super alignment including many major families within the classical ubiquitin superfamily, as well as individual alignments of all newly-discovered β-GF domains. Additionally, we have provided the protein identifier (gi) numbers along with a brief description of all sequences (including organism name and lineage in which the sequence is found) for all identified families of the β-GF.1) last sentence of last paragraph of section "Identification of β-GF domains"Some quantitative data illustrating the comparisons of distributions of previously characterized domains would be desirable. The statement that the majority of conserved lineages has been determined is very strong and depends on the upper level of allowed sequence and structural deviation.We now write, "a significant fraction" instead of majority. We have also referred to a previous work on the matter.2) The reader might be offended to find the definition of domain architectural complexity only in the figure legend of Fig. We have moved the definition of the complexity quotient from the figure legend to the main text.β-grasp families: members, phyletic patterns, and selected alignments. A mega-alignment of several prokaryotic and eukaryotic β-GF lineages and alignments of all newly-identified β-grasp folds and novel ancillary domains are provided along with gi numbers of the sequences collected from iterative database searches and the phyletic pattern of each β-grasp family.Click here for fileNatural classification of the β-GF. Superfamilies/families/subfamilies of the β-GF are grouped according to shared structural and sequence features, indentations represent the inferred hierarchy of evolutionary relationships based on these features. Major shared features of a set of superfamilies are given between dashed lines and labeled with roman numerals. Superfamilies are written in bold text, with families and subfamilies written in bold, italicized text. Phyletic distribution of a family/subfamily are given in parentheses after name. Brief description of functional role is given following phyletic distribution where known. Selected PDB identifiers of solved crystal structures are indented and listed underneath family/superfamily.Click here for file


Ser/Thr/Tyr kinases (STYKs) commonly found in eukaryotes have been recently reported in many bacterial species. Recent studies elucidating their cellular functions have established their roles in bacterial growth and development. However functions of a large number of bacterial STYKs still remain elusive. The organisation of domains in a large dataset of bacterial STYKs has been investigated here in order to recognise variety in domain combinations which determine functions of bacterial STYKs.Using sensitive sequence and profile search methods, domain organisation of over 600 STYKs from 125 prokaryotic genomes have been examined. Kinase catalytic domains of STYKs tethered to a wide range of enzymatic domains such as phosphatases, HSP70, peptidyl prolyl isomerases, pectin esterases and glycoproteases have been identified. Such distinct preferences for domain combinations are not known to be present in either the Histidine kinase or the eukaryotic STYK families. Domain organisation of STYKs specific to certain groups of bacteria has also been noted in the current anlaysis. For example, Hydrophobin like domains in Mycobacterial STYK and penicillin binding domains in few STYKs of Gram-positive organisms and FHA domains in cyanobacterial STYKs. Homologues of characterised substrates of prokaryotic STYKs have also been identified.The domains and domain architectures of most of the bacterial STYKs identified are very different from the known domain organisation in STYKs of eukaryotes. This observation highlights distinct biological roles of bacterial STYKs compared to eukaryotic STYKs. Bacterial STYKs reveal high diversity in domain organisation. Some of the modular organisations conserved across diverse bacterial species suggests their central role in bacterial physiology. Unique domain architectures of few other groups of STYKs reveal recruitment of functions specific to the species. The list of predicted STYKs used for further analysis has been arrived at after careful cross-referencing between the results of these methods as well as manual scrutiny for a variety of factors such as length of the kinase domains and presence of critical functional residues. Kinase catalytic domains of the PKLA were aligned using CLUSTALW. Manual decisions are taken considering the presence/absence of functional motifs and the lengths of putative kinase domains. Those kinase-like sequences without functional residues, such as an aspartate in the catalytic base position, are segregated separately and are not considered for the detailed analysis. Number of such sequences in various genomes is provided in Table We have employed multiple sensitive sequence search and analysis methods PSI-BLAST , IMPALA Domain assignment to the non-catalytic regions of the kinase containing genes has been carried out using the HMM search methods by querying each of the kinase containing sequences against the 6190 protein family HMMs available in the Pfam database . Trans-mThe 3-dimensional structures were superimposed using STAMP .AK performed the computational sequence analysis and modelling. AK and NS conceived the study and participated in its design and coordination. Both the authors read and approved the final manuscript.Data files comprising of the description of protein kinases and homologues encoded in genomes of organisims considered in the current analysis are provided as supplementary information accompanying this article. Each additional data file lists the gene identifiers, length, and domain arrangement of protein kinases and homologues identified in the current analysis.Click here for file


Saccharomyces cerevisiae is one of the best-studied model organisms, yet the three-dimensional structure and molecular function of many yeast proteins remain unknown. Yeast proteins were parsed into 14,934 domains, and those lacking sequence similarity to proteins of known structure were folded using the Rosetta de novo structure prediction method on the World Community Grid. This structural data was integrated with process, component, and function annotations from the Saccharomyces Genome Database to assign yeast protein domains to SCOP superfamilies using a simple Bayesian approach. We have predicted the structure of 3,338 putative domains and assigned SCOP superfamily annotations to 581 of them. We have also assigned structural annotations to 7,094 predicted domains based on fold recognition and homology modeling methods. The domain predictions and structural information are available in an online database at http://rd.plos.org/10.1371_journal.pbio.0050076_01. Saccharomyces cerevisiae proteins into nearly 15,000 distinct domains and then used de novo structure prediction methods together with worldwide distributed computing to predict structures for all domains lacking sequence similarity to proteins of known structure. To overcome the uncertainties in de novo structure prediction, we combined these predictions with data on the biological process, function, and localization of the proteins from previous experimental studies to assign the domains to families of evolutionarily related proteins. Our genome-wide domain predictions and superfamily assignments provide the basis for the generation of experimentally testable hypotheses about the mechanism of action for a large number of yeast proteins.The three-dimensional structure of a protein can reveal much about that protein's evolutionary relationships and functions. Such information about all the proteins in an organism—the proteome—would offer a more global view of these relationships, but solving each structure individually would be a formidable task. In this study, we have parsed all  Saccharomyces cerevisiae domains that were found to have no sequence similarity to any proteins of known structure.Protein three-dimensional structures were predicted for all Saccharomyces cerevisiae is one of the most widely studied organisms, yet a large fraction of its proteins are of unknown structure and/or unknown function. Knowledge of the structure of a protein is critical to understand how it functions, and hence, a complete set of protein structures for yeast is desirable, but difficult to accomplish experimentally.The yeast The accuracy of de novo structure prediction methods, although far from the accuracy of experimental structures, has improved in recent years. The Rosetta de novo structure prediction method –4 is curThere is a rich body of work on the relationship between superfamily  were parsed into structural domains using Ginzu ,19. Ginzhttp://rd.plos.org/10.1371_journal.pbio.0050076_01.Although the confident fold recognition results generated as part of this study are not the main focus of this paper, they provide a wealth of information on proteins for which there are no detectable sequence homologs of known structure. The results for 1,361 domain annotations using fold recognition are detailed in http://wcgrid.org).A total of 4,006 yeast protein domains shorter than 150 amino acids  and not linked to known structures by PSI-BLAST or fold recognition methods were identified by Ginzu: 668 of these contained predicted transmembrane helices and were omitted; the remaining 3,338 domains were folded using the Rosetta de novo method. Ten thousand structure models were generated for each of these remaining 3,338 domains using the Rosetta de novo method ,28 and tPMCM, ranges between 0 and 1 and is an estimate of the probability of the identification of the correct structure superfamily identification. A total of 404 domains in the yeast dataset (see PMCM above 0.8 (considered significant for the purpose of this discussion) and can be found in The 30 representative models for each domain were compared to a database of experimentally determined protein structure domains . Although there is a non-negligible presence of errors in domain parsing and superfamily assignment, our results show that the superfamily assignments generated herein see  should bP(GO|SF), that relate SCOP superfamily (SF) to molecular function, biological process, and cellular component (GO). We also constructed probability distributions, P(SF|D), that give the probability of a given superfamily, given the predicted structures (D), that is derived from the distributions of PMCM for a target, as described in Materials and Methods. These distributions were integrated to determine the degree to which a superfamily prediction is simultaneously compatible with the structure predictions and the functional annotation available for a given protein, using:P is the probability that the domain belongs to SCOP superfamily SF, given the predicted structures, D, and the GO terms, GO, for the protein. The independence assumption underlying There is a strong relationship between the function of a protein and its structural superfamily . Most coP(SF|D)), the GO annotations (P(SF|GO)), and from the two together ), are compared in P(SF|D) (red line) and P(SF|GO) (blue line) are reduced upon integration P (black line), resulting in less ambiguous predictions for many difficult-to-annotate domains. The overall performance for the P over the benchmark set  over 0.8  ≥ 0.8 are correct; five of which (three correct) also have a PMCM of 0.8. The small sample size represented by this dataset makes it difficult to assess accurate upper and lower bounds of the estimated error. We have also generated a much larger dataset  above 0.8 were correct; and 31 of these predictions had both a PMCM and a P above 0.8, and 27 of these were correct. Over all three validation sets, more than 40% of the predictions with a PMCM above 0.8, and more than 75% of the predictions with a P above 0.8, are correct, illustrating the value of data integration in this work.True performance of these technologies cannot be assessed on the benchmark dataset because the domain boundaries of this set are perfect (derived from known structures in the ASTRAL database). A subset of the proteins without links to known structure at the start of this project now have strong homology to a structure that has since been solved, see aset see , as partImportantly, we were able to use these sets of recently solved proteins to better characterize the errors associated with different confidence Ginzu domain predictions. We found that a subset of the incorrect domain parses which significantly diminish the chances of correctly predicting fold and function are easily removed using a simple filter . This domain-prediction filter allows us to recover more-accurate predictions for multi-domain proteins. We were able to classify 50% of the amino acids from the 6,238 attempted ORFs to SCOP superfamilies which is significantly higher than the 35% coverage achieved by a sequence-based hidden Markov model approach .In this section, we discuss several protein complexes with components assigned to superfamilies by both GO-integration and MCM approaches. These predictions and the much larger set of predictions in the database accompanying this paper provide a basis for hypothesis generation and experimental testing, but it must be borne in mind that there is a significant probability that any single prediction is incorrect, as indicated by our estimates of error.The mediator complex, a large complex containing 24 polypeptides , has beePMCM = 0.43; GO-term: transcription from RNA polymerase II promoter; P = 0.83); implying DNA binding. MED4  = 0.98). Several superfamily predictions for this complex (such as hits to superfamilies like spectrin-like and Rossmann folds) are difficult to interpret unambiguously due to the large number of functions compatible with each of these superfamilies, but are not incompatible with DNA-binding functions. Gal11  = 0.92).TIF35 D is a suPMCM = 0.31; GO-term structural constituent of ribosome; P = 0.86), a superfamily involved in RNA binding. We predict that MRPL44  = 0.86). Overall, the structure predictions for these mitoribosome proteins suggest that they belong to superfamilies compatible with known, although highly diverged, components of both the bacterial and eukaryotic ribosome.The mitochondrial ribosome, or the mitoribosome, shares a number of protein components with bacterial ribosomes, but it is believed that the mitoribosomes have comparatively more proteins than their bacterial counterparts; many of the proteins associated with the mitoribosome have no detectable sequence similarity to other mitochondrial proteins . We havet MRPL44 F belongsPMCM = 0.73; GO-term: ATP synthesis couple protein transport; P = 0.82). Inh1 dimerizes and binds to the F1 complex of the ATPase, thereby inhibiting its function [INH1 G is an Afunction ,37. Manyhttp://rd.plos.org/10.1371_journal.pbio.0050076_01. The data will also be made available in other formats upon request.All data are accessible via the Yeast Resource Center (YRC) public data repository  at http:Comprehensive generation of three-dimensional structures with resolution or reliability of those determined by X-ray crystallography or nuclear magnetic resonance (NMR) is currently beyond the capabilities of any protein structure prediction method; these methods can, however, play an important role in generating structural annotations for whole genomes due to the much lower investment of resources required per protein domain. In this work, we have shown that it is possible to: (1) generate protein structure models on a genome-wide scale, (2) automate the assessment of the structure prediction quality, (3) convert the results into pre-existing encodings of structure in the form of SCOP superfamily classifications, and (4) augment the model-based assignment of SCOP superfamily by integrating with pre-existing function, process, and component information encoded in the GO database.We were able to assign SCOP superfamilies to 7,094 of the 14,934 predicted domains in yeast using PSI-BLAST and fold recognition methodology. A total of 4,006 of the remaining 7,840 domains were short enough (less than 150 amino acids) for de novo structure prediction. Of these, 668 were omitted because they contained at least one predicted transmembrane helix. Low-resolution structure models were built for the remaining domains using Rosetta; of these, 404 were assigned to superfamilies with confidence using MCM, and an additional 177 were assigned with confidence after integrating with GO process, component, and function annotations.http://wcgrid.org), innovative database architecture [A significant challenge in carrying out this work was the magnitude of the computation required for generating de novo structure predictions for large numbers of domains. Robust and fast methodology, efficient data storage, analysis tools, and data organization were required. Our use of distributed computing , the length of the two proteins involved, LAstral and Lpredicted, the CO of the region of the predicted structure that was superimposable on the experimental structure, and the degree to which Rosetta converged during the generation of the set of predicted conformations . The general formula for the confidence functions is given in a, b, c, d, and the constant C) for the three models described in the following paragraph are presented in The MCM estimates the probability that the MAMMOTH match between predicted structure and the ASTRAL domain (see previous section) has identified the correct superfamily and is based on the closeness of match  as follows. Each superfamily is initially assigned a probability corresponding to the maximum PMCM value for that superfamily over the top five PMCM values for all predicted conformations for the query protein; probabilities less than 0.2 are set to zero. If the sum of the raw probabilities is greater than 0.8, they are scaled linearly so that the sum is 0.8. Because of the uncertainties of de novo structure prediction, these scaled probabilities, Pscaled(SF|D), are then linearly combined with the background superfamily distribution, P(SF)  :P(SF|D), are guaranteed to have non-zero probabilities for every superfamily, and to sum to 1. The background distribution P(SF) ensures that (1) we do not disregard useful functional information at the integration with GO stage and (2) that we do not over interpret the confidence values derived from the benchmark training set.The final distributions, P of a superfamily, SF, given both protein structure prediction, D, and GO annotations, GO, using Bayes' rule and the assumption that P ∼ P(GO|SF)*P(D|SF):P(D|SF) via We obtain P(SF) and P(D) cancel. P(SF|D) is computed as described in the previous section, and P(GO|SF), P(GO), and P(SF) are computed from proteins in the PDB that are annotated with GO function, component, or process and also classified in SCOP. To deal with cases in which there is a single function annotation for a given superfamily, we allow for the possibility that the uniqueness of this mapping is due to under-sampling of superfamily space (as represented by the PDB) or function space (as represented by GO) by adding pseudo counts distributed according to the background superfamily distribution, Pastral95(SF), computed from ASTRAL 1.67 culled so that no sequences are more then 95% identical.After substituting M (a regularization parameter controlling the relative contribution of our pseudo-counts) was estimated by carrying out function assignment given the superfamily over the benchmark set: we chose M to minimize the classification error estimated using 10-fold cross-validation. The overall procedure was relatively insensitive to the value of M ranging from one to ten with an optimal value of four. The P(SF|GO) are too diffuse for confident superfamily prediction from GO annotations alone, hence the integration with the structure prediction data is critical for accurate superfamily predictions.The parameter P for any given protein domain is normalized to sum to one; thus confident assignments are not made when there are strong matches to more than one superfamily.Finally, the sum of e-value less than 1 × 10−3. We also require that the shorter of the two sequences is more than 80% of the length of the longer one, and that 60% or more of the predicted domain is aligned with the ASTRAL domain. These domains are part of an ongoing project in which we predict structures for over 150 genomes; although domains with any homology to known structures are excluded, a number of structures have been solved and classified in SCOP during the 18 mo the project has been running. The scope of this separate project prohibited us from carrying out fold recognition calculations on these domains, and since domains that can be assigned using fold recognition methods will on average have higher MAMMOTH structural similarities to known structures than domains that cannot be assigned, results from this dataset represent an upper bound on performance on the dataset in this paper.The performance of the MCM and the GO integration was evaluated on two independent datasets. The first dataset, from HPF project, consists of 768 predicted domains that now have a homolog with a known structure that is classified in SCOP 1.69. The homologs were identified by blasting predicted domains against all sequences from ASTRAL 1.69 and selecting those with a PSI-BLAST The second dataset was generated the same way the HPF set was generated, but limited to yeast domains. The proteins from which these domains are derived have been subjected to fold recognition and hence give a better estimate of the true performance. This dataset is, however, too small for statistically significant conclusions to be made.Based on inspection of the results on the HPF dataset, domains from predicted two-domain proteins are excluded if both the domains are predicted using less-confident methods , or if the domain under consideration is an MSA domain regardless of the neighboring domain type. A large fraction of these proteins have single domains, and correct superfamily matches are quite unlikely when models are only generated from domain fragments.The generation of structure predictions was divided into three completely automated steps: pre-processing, production (the running of Rosetta), and post-processing . The pre-processing protocol includes domain prediction, prediction of secondary structure, disordered regions, trans-membrane helices , and sigTable S1All 14,934 domains predicted from the 6,238 sequences are presented in detail.(4.7 MB PDF)Click here for additional data file.Table S2The most confident predictions using the MCM are listed.(133 KB PDF)Click here for additional data file.Table S3The most confident predictions using the GO-integration strategy are listed.(77 KB PDF)Click here for additional data file.Table S4Best predicted structure—the best predicted structure by RMS among the 1,000 created; Top5 is the cluster center from the five largest clusters; Best Match—the best domain match from all 30 cluster centers.(476 KB PDF)Click here for additional data file.


The PD-(D/E)xK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold.The RecC C-terminal region can be divided into an alpha/beta domain and a smaller alpha-helical bundle domain. Here we show that the alpha/beta domain is homologous to the RecB nuclease domain but lacks the features necessary for catalysis. Instead, the domain has a novel function within the nuclease superfamily – providing a hoop through which single-stranded DNA passes. Comparison with other structures of nuclease domains bound to DNA reveals strikingly different modes of ligand binding. The alpha-helical bundle domain contributes the pin which splits the DNA duplex.The demonstrated homology of RecB and RecC shows how evolution acted to produce the present RecBCD complex through aggregation of new domains as well as functional divergence and structural redeployment of existing domains. Distantly homologous nuclease(-like) domains bind DNA in highly diverse manners. The largest evolutionary superfamilies of proteins cover such a large range of sequence space that the relationships shared by members may not be apparent by standard means of sequence comparison, and hence are often only recognized after structural determinations. Such has frequently been the case for the PD-(D/E)xK superfamily of nucleases. Within the superfamily, structures were first obtained for four restriction enzymes, of such diverse sequences that they were initially assumed not to share homology (reviewed in ). Since S. cerevisiae RPB5, an RNA polymerase subunit, where evidence suggests that it functions in protein-protein interactions [The unifying catalytic site characteristic of the superfamily is the presence of one or more catalytically essential divalent cations ,7. The cractions .Although extremely diverse in structure and sequence, modern sequence comparison methods have played their part in elucidating the full range of PD-(D/E)xK superfamily members ,13-15. NHere we show that the C-terminal region ('domain 3') of RecC can actually be dissected into two domains, the first of which is clearly related to PD-(D/E)xK superfamily nuclease domains  and particularly to the corresponding domain of RecB. The nuclease-like domain of RecC is inactivated and therefore possesses not even the metal-ligating residues of the PD-(D/E)xK motif. Instead, it carries out a function not hitherto observed in the superfamily, providing an aperture through which one strand of newly split DNA duplex is fed. Comparisons show that nuclease(-like) domains are extraordinarily versatile in their mode of interaction with duplex DNA. Characteristics of the RecC nuclease-like domain show that RecB and RecC share a common ancestor and reveal how evolution has progressed by sequential addition of domains to the C-terminus, as well as by altering function of, and repositioning of, existing domains.Domain 3 of RecC has been described as being of novel fold . StructuThe nuclease fold common to λ-exonuclease, RecB and now RecC is found in a wide variety of exo- and endonucleases, from restriction enzymes to Holliday junction resolvases, and enzymes of DNA repair . Within Interestingly, comparison of the nuclease domains of RecB, RecC and λ-exonuclease shows that the Rec subunits clearly share a more recent ancestor than the common ancestor of all three structures. As Figures As mentioned, nuclease domains as represented in the present PDB are extremely diverse in sequence but share conserved residues that bind essential metal ions and are almost invariably catalytically active. The recognition of the third domain of RecC as an inactivated nuclease domain highlights a wholly unexpected new function for a non-catalytic but clearly nuclease-like domain. As shown in Fig E. coli RecC the connection is a minimal β-turn and connections in other RecC sequences are also very short  in the results of the first iteration. In contrast, using an e-value cut-off of 0.0001 four iterations are required before RecC sequences, including that of E. coli RecC, appear among the significant hits. While the BLAST alignments centred on the helicase(-like) domains the C-terminal nuclease(-like) domains were sometimes matched, although PSI-BLAST runs of the nuclease domain of RecB failed to hit the nuclease-like domain of RecC, and vice versa. Similarly, structural comparisons show that both helicase domains and both α-helical insert domains of RecB are more similar to their counterparts in PcrA than to the corresponding RecC domains (not shown). Nevertheless, the clear homology of the RecB and RecC nuclease(-like) domains, evident in their common three α-helical bundle (see above) strongly suggests that RecB and RecC share a more recent common ancestor than they have in common with other extant helicases. How then to explain the apparently closer relationship of RecB with PcrA than with RecC? As was proposed for the nuclease(-like) domains (see above) it seems like the dramatic functional differences between corresponding RecB and RecC domains are responsible. As discussed above, the RecC nuclease-like domain is significantly shorter than the RecB nuclease domain in two key regions, each associated with its new role as provider of an ssDNA hoop. Thus, it seems plausible that the maintenance of helicase activity by the helicase domains of PcrA and RecB is responsible for their apparently closer relationship, the structural changes accompanying evolution of the helicase-like domains in RecC for new roles having obscured their more recent shared ancestry with RecB.The observation of inactivated helicase-like domains in RecC was not considered reason enough to propose the existence of homology between RecB and RecC extending over their whole length . Indeed,e domain , appearsThe recognition of homology between RecB and RecC, and the dissection of their domains leads to an interesting comparison with PcrA. In PcrA, a duplication of an ancestral RecA-like domain, already containing an all α-helical insert domain, is evident . In RecBThere is an interesting parallel to be drawn between RecBCD and AddAB , a different DNA repair system found in Gram positive bacteria where RecBCD is lacking . Protei


Dishevelled prevents β-catenin degradation by interfering with the interaction of β-catenin with the degradation-mediating Axin-APC-GSK3β complex. This interference leads to an accumulation of cytoplasmic β-catenin, which enters the nucleus and interacts with transcription factors that induce expression of Wnt-target genes. Axin, as a component of the degradation-mediating complex, is a potent negative regulator of Wnt signalling, whereas Dishevelled is a potent activator. Both Dishevelled and Axin possess a DIX (Dishevelled/Axin) domain, which mediates protein-protein interactions, specifically homodimerization.The An evolutionary trace analysis of DIX domains identified conserved residues which, when mapped onto the crystal structure of the Axin DIX domain and a comparative model of the Dishevelled DIX domain, allow their categorization as residues of either structural or functional importance. We identify residues that are structural and functional determinants of the DIX domain fold, as well as those that are specific to homodimerization of Axin and Dishevelled.This report provides the first explanation of the mutant phenotypes caused by non-synonymous substitutions in the Dishevelled and Axin DIX domain by correlating their presumed functional significance with molecular structure. Signalling by members of the Wnt family of secreted glycoproteins relies on dynamic interactions between protein complexes to regulate transcription, the cytoskeleton and cell adhesion ,2. BindiIn the 'canonical' pathway, Wnt proteins regulate transcription of specific genes by modulating the amount, activity and intracellular location of β-catenin, a multi-domain protein with a role in linking Cadherins to the cytoskeleton, in addition to being an effector of Wnt signalling . In the The central role of Dishevelled in Wnt signalling and the key role of Axin in the activity of β-catenin suggest that the interaction between these two proteins represents a central event for Wnt activity. The interaction between these two proteins is mediated for the most part through their DIX (Dishevelled/Axin) domains, although other regions of both proteins are also involved ,6,8. A tThe DIX domain is a region of approximately 83 to 85 residues in length located at the N-terminus of Dishevelled and at the C-terminus of Axin and DIXdc1 Figure . While tOnly a limited number of functionally important amino acids in DIX domains have been identified and additional single or multiple substitutions may shed light on the precise function of this domain. Here we have performed a sequence-structure analysis of the DIX domain of Axin and Dishevelled using the evolutionary trace analysis approach first described by Lichtarge et al. , to idenA comparative model of the human Dishevelled 1 DIX domain, residues M1 to E85, was created using Modeller 9v1  based onOne hundred and twenty sequence homologues of the human Dishevelled 1 DIX domain were identified using BLASTp with the database search parameter set to non-redundant protein sequences. Of these, 63 were retained for evolutionary trace analysis  and the comparative model of the Dishevelled DIX domain. Based on their location in the relevant structure, the trace residues were classified as either (1) conserved and buried, (2) conserved and surface exposed, (3) class-specific and buried or (4) class-specific and surface exposed. Class-specific residues are residues that are conserved only in a subset of sequences, such a subset being defined as a class. Residues were classified as buried if less than 30% of their side chain is solvent accessible.Rattus norvegicus (1WSP) was the only structural homologue of the human Dishevelled DIX domain identified with certainty by the homology recognition server FUGUE (Z-score 19.18). The R. norvegicus Axin and the human Dishevelled DIX domain share 30% sequence identity. A sequence-based secondary structure prediction of the human Dishevelled DIX domain showed that the position and identity of secondary structural elements match those of the Axin DIX domain crystal structure . Given t shown; , and theThe DIX domain has a typical β-grasp fold characteristic of the ubiquitin superfamily. A search of the Protein Databank using the fold comparison server DALI identified PB1 domains as structurally similar to DIX domains, sharing similar topology and, after examination of their dimeric structures, similarity to the putative Axin DIX homodimer evident in the crystal structure of that domain . The PB1Ciona intestinalis, still shares 45% sequence identity with human Dishevelled. This is also evident in Additional file In order to determine the possible structural or functional role of specific residues in the DIX domain, sequences of DIX domain-containing proteins were aligned and the observed sequence conservation mapped onto the DIX domain structures. The alignment of available Axin, Dishevelled and DIXdc1 proteins shows that there are only few residues that are absolutely conserved . Somvelled 2 ,6,8. In The sequence-structure based analysis presented here identifies a cluster of functionally significant residues located on loop L4 and strand β4 that contain most of the conserved residues in DIX domains. We identify residues that are structural and functional determinants of the DIX domain fold and residues that likely mediate specific homotypic interactions between Axin DIX domains and Dishevelled DIX domains. Based on the domains structural homology to PB1 domains, which are known dimerization modules, and the observed crystal contacts between chains in the asymmetric unit of the Axin DIX crystal structure, it is likely that the residues clustered on loop L4 and strand β4 constitute the dimerization interface of DIX domains in general. Existing mutagenesis data identified several residues of functional importance at this interface, supporting this conclusion. We predict that several other residues are also important to function. Investigating these should shed more light on the precise role of DIX domains as dimerization modules.MTE design the study, performed the modelling, analysis and drafted the manuscript. AMA critically reviewed the manuscript and provided general support and advice throughout the study. All authors read and approved the final manuscript.Accession numbers of sequences used in this study. All accession numbers are those of the GenBank database.Click here for fileStructure-based multiple-sequence alignment of DIX domains from Axin, Dishevelled and DIXdc1. Residues that are absolutely conserved have a black background, those that are conserved in a majority of sequences are shaded grey or light grey. The secondary structure assignment of the Axin 1 Rattus norvegicus crystal structure 1WSP is shown above the alignment. # coloured cyan are sites of non-synonymous amino acid substitution in Axin that have no effect on or only diminished ability to dimerize and * coloured cyan are unable to dimerize [Rattus norvegicus Axin 1 and for Homo sapiens Dishevelled 1 (DSH1) are given. Each protein sequence is labelled with the protein name followed by the name of the species the sequence is from. In the case of Axin and Dishevelled, the numbers after the protein name, where present, denote orthologs. DSH, Dishevelled; XDSH, Xenopus Dishevelled; DIXdc1, DIX domain-containing 1.dimerize . * coloudimerize , whereasdimerize ,30. The Click here for fileDendrogram of the DIX domain containing proteins. Partitions P01 to P10 are shown as vertical red lines. Evolutionary time-cut off increases from P01 to P10. The Rattus norvegicus DIX domain used to create the comparative model of the human Dishevelled 1 DIX domain are indicated with cyan coloured labels 1WSP and DIX, respectively. Yellow shaded area contains members of the Axin family, the orange shaded area members of the Dishevelled family.Click here for file


These observations provide accurate constraints for models of fibrillin organization within the 10–12 nm microfibrils and provide further molecular insights into how Ca2+ binding influences the intermolecular interactions and biomechanical properties of fibrillin-1.The fibrillins and latent transforming growth factor-β binding proteins (LTBPs) form a superfamily of structurally-related proteins consisting of calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with 8-cysteine-containing transforming growth factor β-binding protein-like (TB) and hybrid (hyb) domains. Fibrillins are the major components of the extracellular 10–12 nm diameter microfibrils, which mediate a variety of cell-matrix interactions. Here we present the crystal structure of a fibrillin-1 cbEGF9-hyb2-cbEGF10 fragment, solved to 1.8 Å resolution. The hybrid domain fold is similar, but not identical, to the TB domain fold seen in previous fibrillin-1 and LTBP-1 fragments. Pairwise interactions with neighboring cbEGF domains demonstrate extensive interfaces, with the hyb2-cbEGF10 interface dependent on Ca The 10–12 nm fibrillin microfibrils of the extracellular matrix play important roles in both elastic and nonelastic tissues. During elastogenesis, they provide a scaffold for the deposition of tropoelastin and form the periphery of the mature elastic fiber. In nonelastic tissues, such as the ciliary zonule of the eye and at basement membranes, they have an anchoring function and provide tissues with tensile strength . In addiAlthough it has been known for several years that fibrillin is the main structural component of the microfibrils , the preThe molecular organization of the fibrillins, and the related LTBPs, is dominated by calcium-binding epidermal growth factor-like (cbEGF) domains . These a2+ binding by both of the fibrillin-1 hyb-cbEGF domain pairs show that the cbEGF domains in this context bind with high affinity, with Kd values in the low nanomolar range. As in the case of TB-cbEGF domain pairs, this is due to interdomain hydrophobic interactions stabilizing the Ca2+ binding pocket of the cbEGF domain. These data can be used to extend our knowledge of fibrillin structure and provide new constraints for models of microfibril organization.Here we present the X-ray crystal structure of a cbEGF9-hyb2-cbEGF10 triple domain fragment from fibrillin-1, providing the first high-resolution structure of a hyb domain found in the fibrillin/LTBP protein family. We show that these highly disulphide-bonded domains are structurally related to both the TB and EGF domains, as suggested by previous sequence data , with an2 in order to saturate the N-terminal cbEGF9 Ca2+ binding site, which is likely to have a relatively low affinity for Ca2+ based on previous work .To characterize fully the architecture and interdomain interactions of the hyb-2 domain and its adjacent cbEGF domains, a crystal structure of the fibrillin-1 construct cbEGF9-hyb2-cbEGF10  was deteous work . Crystalk , three main-chain carbonyls , and a single water molecule , three main-chain carbonyls , and a single water molecule  is also seen in the hyb2 domain in the position corresponding to the cis-proline observed previously in the TB4 structure (Pro1573)  . The majPro1573) C.In both the TB and hyb domains, the first two of the cysteines (C3 and C4) in the central α helix form disulphide bonds with cysteines toward the N-terminal part of the domain near the hydrophobic core. In the TB domains, a third disulphide is formed between C5 in the central α helix and C8, which is positioned between the second α helix and the final two strands of β sheet. The hyb2 structure shows a rearrangement of disulphides toward the C-terminal part of the domain so that the cysteine corresponding to C8 in the TB domain forms a disulphide bond with the second to last residue of the domain. This is likely to stabilize the C-terminal β sheet of the hyb domain and changes the arrangement of disulphides from C1-3, 2-6, 4-7, 5-8 in the TB domains to C1-3, 2-5, 4-6, 7-8 in the hyb domain E.2+ plays an important role in the formation of interfaces within cbEGF-cbEGF and TB-cbEGF pairs, and confers a rigidity to these structures . In addition, the Ca2+ affinity of a cbEGF9-hyb2-cbEGF10 construct , suggested that the addition of a cbEGF to the N terminus of the hyb2 domain had no significant effect on the Ca2+ affinity of cbEGF10, and that the high Ca2+ affinity of this construct is due entirely to hyb2-cbEGF10 interfacial interactions.Assays of the hyb2-cbEGF10 pair with the chromophoric Caonstruct  was meas2+ binding by the hyb1-cbEGF pair of fibrillin-1 was studied. Chelation competition assays of the native hyb1-cbEGF1 domain pair were not possible due to the insolubility of this fragment. Not unexpectedly, this insolubility was found to be due to the presence of a free thiol, as determined by assays with Ellman's reagent (data not shown), which became reactive when the protein was dissolved in buffers at physiological pH. Ca2+ binding was therefore measured for a hyb1-cbEGF1 construct containing a Cys204Ser substitution, which removed the free thiol previously shown to be involved in intermolecular interactions . Molecular modeling of hyb1, based on the hyb2 structure, shows that the extra cysteine of hyb1 is likely to be positioned at the N terminus of the central α helix of the domain in a solvent-exposed position . Chemical shift changes were seen for both cbEGF and hyb domain residues, indicating that interface formation in the hyb2-cbEGF10 pair is Ca2+-dependent as seen previously in TB-cbEGF domain pairs  and heteronuclear single quantum coherence (HSQC) spectra of in pairs .2+ concentrations (∼1.5 mM free Ca2+), the region around hyb2 would be near linear with N and C termini emerging at opposite ends of the fragment. Based on our current knowledge of cbEGF, TB, and hyb domain structures  of hyb2 separates the N-terminal TB-like region from the C-terminal cbEGF-like region. Under tension, this loop could allow the disruption of the hyb-cbEGF interface, extending the length of the structure by approximately 3.5 nm. Recoil of the system would be achieved by the reformation of the hyb-cbEGF interface, which is stabilized by the binding of Ca2+ binding to cbEGF10. Such dynamic behavior could facilitate the structural role of fibrillin in connective tissues that are subjected to high tensional forces.Our study has provided the first high-resolution structure and possible evolutionary origin of a hybrid domain, one of the major disulphide-rich domains of the fibrillin/LTBP superfamily. Overall the fibrillin-1 hyb2 domain has a globular shape, resembling a TB domain, and is involved in extensive interactions with its neighboring domains, cbEGF9 and cbEGF10. A mechanism for the evolution of the hybrid domain as a fusion of TB and cbEGF domains was first proposed by 2+ affinity of the TB4-cbEGF23 pair (Kd = 16 nM) in this fragment was in contrast to previous observations of a TB6-cbEGF32 fragment (Kd = 1.6 mM), suggesting that Ca2+ affinities across the molecule could vary substantially. This was found to be the case in a study of all fibrillin-1 TB-cbEGF domain pairs, where it was also shown that Ca2+ affinity could be correlated with the degree of interdomain interaction. Given the relatively high affinities seen for all the TB-cbEGF pairs except TB6-cbEGF32, it was suggested that these regions of fibrillin form rigidified, linear structures at Ca2+ concentrations found in the extracellular matrix  of cysteine-free sequence. LTBPs 1, 3 and 4 have been shown to bind covalently the latency-associated peptide of TGFβ through the formation of an intermolecular disulphide bond, with the interaction mediated by the rearrangement of a disulphide bond in the second TB domain of the LTBPs. The structural basis of this rearrangement has been well-characterized and is due to the presence of a two amino acid insertion between C6 and C7 of the TB domains, leading to the increased solvent accessibility of the C2-C6 disulphide bond. In the fibrillin-1 hyb2 structure, no comparable arrangement of disulphides was observed, suggesting that this domain is unlikely to be involved in disulphide rearrangements. In contrast, domain hyb1 has been shown to be involved in the formation of intermolecular disulphides as a result of the presence of a conserved 9th cysteine . Our mod2+-saturated cbEGF9-hyb2-cbEGF10 structure presented in our study shows that domains cbEGF9 and cbEGF10 pack against domain hyb2 to form a near-linear structure. Ca2+ binding data for the hyb2-cbEGF10 pair shows that an extensive interface is formed between these domains, as seen previously in TB-cbEGF pairs where specific hydrophobic contacts between domains were found to influence Ca2+ affinity . Constructs corresponding to protein fragments hyb1-cbEGF1 (Arg179 to Glu287), cbEGF9-cbEGF10 (Asp807 to Leu951), and hyb2-cbEGF10 (Glu847 to Leu951) were amplified from a human fibrillin-1 cDNA , 200 mM NaI, and 16% PEG 3350. All crystals were washed with mother liquor supplemented with 25% (v/v) glycerol before storage in liquid nitrogen. Native X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF). Initial phases were determined from SAD data collected in-house using a Bruker MicroStar Smart 6000 CCD X-ray generator, with signals from both sulfur and calcium . The structure was rebuilt and refined using Coot  were titrated with Ca2+ stock buffer  in the presence of Quin-2. All titrations were performed with 20 – 30 μM chelator and 20 – 30 μM protein at 23 ± 2°C using a Shimadzu UV mini 1240 spectrophotometer to monitor absorbance at 263 nm. Background Ca2+ levels in the protein-containing samples were determined by extrapolating the initial linear region of each titration curve back to the y-axis. The initial Ca2+ concentration of each curve was adjusted so that the y-intercept was equivalent to that of a “chelator-only” control. The Kd value for Quin-2 was determined independently from chelator only control experiments; a Kd value of 0.35 μM for Quin-2 was obtained. Free Ca2+ concentration at each titration point was calculated by the Newton-Raphson method, and dissociation constants were calculated by least-squares fitting to the data, as described previously using in-house software (d values were estimated from at least four repeats of each assay.Caeviously . To minisoftware . Errors