This paper is an attempt to trace the evolution of the ribosome through the evolution of the universal P-loop GTPases that are involved with the ribosome in translation and with the attachment of the ribosome to the membrane. The GTPases involved in translation in Bacteria/Archaea are the elongation factors EFTu/EF1, the initiation factors IF2/aeIF5b + aeIF2, and the elongation factors EFG/EF2. All of these GTPases also contain the OB fold also found in the non GTPase IF1 involved in initiation. The GTPase involved in the signal recognition particle in most Bacteria and Archaea is SRP54.1) The Elongation Factors of the Archaea based on structural considerations of the domains have the following evolutionary path: EF1→ aeIF2 → EF2. The evolution of the aeIF5b was a later event; 2) the Elongation Factors of the Bacteria based on the topological considerations of the GTPase domain have a similar evolutionary path: EFTu→ IF→2→EFG. These evolutionary sequences reflect the evolution of the LSU followed by the SSU to form the ribosome; 3) the OB-fold IF1 is a mimic of an ancient tRNA minihelix.The evolution of translational GTPases of both the Archaea and Bacteria point to the evolution of the ribosome. The elongation factors, EFTu/EF1, began as a Ras-like GTPase bringing the activated minihelix tRNA to the Large Subunit Unit. The initiation factors and elongation factor would then have evolved from the EFTu/EF1 as the small subunit was added to the evolving ribosome. The SRP has an SRP54 GTPase and a specific RNA fold in its RNA component similar to the PTC. We consider the SRP to be a remnant of an ancient form of an LSU bound to a membrane.This article was reviewed by George Fox, Leonid Mirny and Chris Sander. This study is on the origin and evolution of the ribosome Large Subunit (LSU) and Small Subunit (SSU) as reflected in the structure of the universal ribosomal translational GTPases. This continues an earlier study  on the oet al. [Recent papers by Bokov and Steinberg , Hury etet al. , and Foxet al.  considerThe translational GTPases are the Bacteria elongation factors, EFTu, EFG, and the initiation factor, IF2, and their archaeal homologs, the EF1, EF2, aeIF5b and aeIF2. There are also two additional GTPases associated with transmission of the translated peptide chain from the ribosome to the membrane, the Ffh/SRP54 as part of the signal recognition particle (SRP) and the GTPase, FtsY/SR-alpha, which is attached to the cell membrane.The initiation factors are involved with bringing the f-Met-charged tRNA to the SSU ribosomal subunit and the joining of the SSU and LSU ribosomal subunits. The elongation factors, EFTu/EF1, bring the next charged tRNA to the ribosome and interact primarily with the large subunit, while EF2/EFG initiate the elongation cycle involving the PTC peptide bond formation along with the allosteric motion of the whole ribosome.EF1, aeIF5b, aeIF2 and EF2 from Archaea and their bacterial counterparts, EFTu, IF2 and EFG, are multidomain proteins. They all contain two homologous N-terminal domains: a GTPase or G-domain, followed by an OB-domain [In addition to the common first two domains, archaeal EF1and bacterial EFTu have a third C-terminal OB-domain (yellow) in common. IF2 and aeIF5b have two additional domains linked by long helices. The domain three of the bacterial IF2 is a simple alpha beta sandwich, while domain four is another OB beta barrel, similar to IF1 and to the second domain of EFTu/EF1.5P21[These translational proteins' G-domains are both structurally and functionally related to a larger family of GTPase G proteins . All con5P21). The seComparison of the other translational protein G-domains with the structural topology ofRas or EFTu allows the identification of their non-core differences. For example, IF2 and EFG contain a number of non-core modifications  protein. This protein has two domains, G domain (GTPase) and an N-terminal domain, a four-helical bundle. The G-domain belongs to the SIMBI class of the P-loop GTPases . The arcAll seven of the translational GTPases displayed in Figure More recently the OB fold has been reviewed by Arcus describing the structure and function of OB fold domains . Among tThere is a weak sequence similarity between the OB folds of EFTu and EF1. However there are no significant sequence similarities among the other GTPase OB second domains. There is a 14-15 amino acid insert at the very start of the OB second domain in EFG that has no equivalent in the archaeal EF2. A pure structural comparison between the bacterial and archaeal homologs of these OB-domains displays considerable similarity. For example the structures of the three bacterial translational GTPase OB second domains show remarkable similarities, see Figure Interestingly the structure similarity of these proteins' second domains carries over to the fourth domain of the IF2's and the entire structure of the IF1's. In the two latter cases the OB-domain likely interacts directly with either the tRNA or the active site of the small ribosomal subunit mRNA . In the As noted above there are very similar OB-domains found in a wide range of protein families, including the translation-related ribosomal protein, S1, containing six such domains. As with EFTu/EF1 and IF2, the second OB-domain of S1 has been shown to directly bind single-stranded RNA as it delivers transfer-messenger RNA (tmRNA) to stalled ribosomes . This, cThere are additional small beta barrel OB-like domains in the EFTu/EF1 and EFG/EF2 homologs as their third domain. These have less bacterial-to-archaeal sequence similarity than the second domains, but with evidence of at least two significant taxonomic domain blocks. A curious fact is that these third domains of EFTu/EF1 and EFG/EF2 are wound in the reverse direction to their common second domains. The reversed OB fold of this third domain interacts with the doubled stranded RNA of the CCA arm of the tRNA .-domains [Connected to the bacterialIF2'sOB second domain via a six-turn helix is a small alpha beta domain that appears to make contact with both the G-domain and the OB second domain. Then there a fourth domain with an OB-fold, connected by a long helix. The latter has a set of loops at the far extremity of the protein that, like other OB -domains , may makMoving beyond the common G-domains and OB-domain of the EFG/EF2 proteins, there are three more domains. In the bacterial case these form a structure spatially similar to a tRNA . The strIn an earlier paper , a modelet al., "The sequence similarity between Ffh and FtsY and the high number of known sequences allow us to perform phylogenetic studies on the SRP-GTPase family. These suggest that the SRP-GTPases might have evolved earlier than the small GTPases" [The current analysis of the translational GTPases, including the SRP and SR GTPases, provides additional support for such a model. It was noted by Montoya GTPases" . One conGTPases" . They weThe common ancestor of the translational GTPases began with a Ras-like GTPase to which an OB fold was joined. This ancient elongation factor was the precursor to EFTu/EF1. The ancestor of the EF2/EFG and IF2 would have come later, evolving from the first two EF1/EFTu domains as the SSU mRNA complex began to interact with the LSU peptidyl transfer center. This would have been coordinated with the evolution of the full tRNA structure. The evolution of the molecular mimicry by EFG of the EFTu tRNA complex, as noted earlier by Fox and Naik , reflectThe IF1 as an OB fold is a small protein (70 to 100 amino acids) and binds to a single stranded RNA. This fold is found in a number of ribosomal proteins. The extant IF1 as a single OB fold appears to block the A-site, forcing initiation to begin in the P-site. Thus this OB fold appears capable of mimicking the anticodon stem loop of an ancient tRNA. An original form of the IF1 OB fold could have been used to modulate peptide formation at the proto-PTC site of the evolving large ribosomal subunit. A later fusion with a Ras-like GTPase then led to the formation of EFTu and the controlled delivery of the activated mini tRNAs to the PTC of the evolving LSU.One further consequence of this evolution of the ancestor of EFTu/EF1 is that it must have been connected with the evolution of the transfer RNA itself. In a previous paper  on the eThe OB fold may reflect this evolution of the ancient tRNA as the OB fold can be viewed as a molecular mimic of the anticodon stem of the ancient tRNA, e.g., IF1. The OB fold may also have interacted with the CCA end of the early minihelical tRNAs, as in the extant EFTu. The third domain of EFTu, the reverse OB fold, would have been added to interact with the helical portion of the ancient tRNAs for stabilization purposes. These interactions are seen in the extant EFTu tRNA complex .The Archaea, as has been noted, have an initiation factor IF2 complex that has two separate protein subunits: the gamma composed of a G-Domain and two OB folds, and a second, alpha, subunit composed of a three-domain subunit similar to those of the three C-terminal domains of the bacterial EFG Fig. . The ArcThe universal sequence blocks found in the translational G-domains imply that modern Bacteria and Archaea  clearly had a common ancestor. Yet the phylodomain-specific blocks imply that the two groups each derive from single, phylodomain-specific types that came into existence some point long after that common ancestor with a fully functional translational system. Why only two basic types? One simple explanation proposed is a major evolutionary bottleneck, drastically limiting the progenitors of modern cell domains .Another hypothesis to consider in the evolution of the translational apparatus is that the earliest translational protein precursors were conglomerates of peptides that only later were amalgamated into a single sequence of amino acids. For example, the universal sequence blocks of the G-domains may be remnants of an earlier peptide form of these proteins. The OB-domain is an obvious example as it can be assembled from multiple copies of such a short peptide that could form a stable beta hairpin. The idea of short peptides interacting with RNA at a very early time is consistent with the proposal that the earliest form of the proto-ribosomal peptidyl transfer center was composed of a few RNA helices stabilized on a peptide membrane .The evolution of the extant ribosome-associated GTPases would have begun with a fusion of an OB fold to a Ras-like GTPase whose function was to transport aminoacyl ancestral tRNAs to the membrane-associated self-folding PTC RNA. The initiation and elongation factors evolved from this EFTu/EF1 by adding modules that allowed these GTPases to interact with the SSU. This is consistent with our original hypothesis that the PTC domain of the ribosomal LSU evolved first.et al. [et al. [The various sequence databases  and structural databases (RCSB and PDB) were searched for a wide range of bacterial and archaeal representatives of the translational GTPase and related proteins. These were examined for their range of variation, and representatives selected. Then using standard sequence comparison methods  and struet al. . In addi [et al. . The ide [et al.  and PyMo [et al. , both foThe authors declare that they have no competing interests.TFS designed the study, performed the research and helped to draft the manuscript. HH designed the study, performed the research and helped to draft the manuscript. All authors read and approved the final manuscript.(1) This manuscript is a significant contribution to the study of ribosome evolutionary history by providing strong evidence for the relative age of the main P-loop GTPases using comparisons of structures and sequences of the various molecules under consideration. It is not clear why EF-P whose structure closely resembles eIF5A  is not considered or even mentioned. It is especially interesting to learn that the GTPase Domain produces recognizable sequence blocks, which suggests largely divergent evolution. With regard to these blocks, it would be appropriate to add the sequence alignments underlying Figure Authors' response: We have focused this paper only on those ribosomal GTPases that are universal both to Bacteria and Archaea. EF-P and eIF5A do have an OB fold similar to IF1 and the GTPase second domains, thus we have added them to the list in our discussion on the OB folds.Minor points (suggested but not required revisions):1) Suggest rotating aeIF2 alpha image in Figure  Suggest Authors' response: This would have required a complete redo. We decided it was only a suggestion.2) In Figure  In FigurAuthors' response: Done(3) In the third paragraph of Background section initiation factor text is confusing- suggest ".... EF-Tu, EFG, IF2, and their archaeal homologs EF-1, EF2, aeIF5b and aeIF2."Authors' response: Rewritten as suggested.(4) In the fourth paragraph of Background section the phrase "while EF2/EFG initiate the elongation cycle via the PTC peptide bond formation." Is unclear and should be rephrased.Authors' response: We rephrased to clarify.(5) In the sixth paragraph of Background section suggest revised wording "".... similar to the second domain of EFTu/EF1 and IF1." In addition, the phrase "not shown" removed because it is not clear what is not shown.Authors' response: Done.(6) In the fifth paragraph of the Results section "with no equivalent bacterial segments" is not true of the second block where the bacterial segment although much smaller than the archaeal segment is nevertheless of similar size as that found in Bacterial EFTU/EF1 and therefore is probably not appropriately referred to as "apparently almost complete deletion". Suggest saying that first has no equivalent and then discussing second separately.Authors' response: Rewritten as suggested.(7) In the first paragraph of the Discussion section the following language might be better--""This RNA IS ENVISIONED AS membrane-bound structure catalyzed the peptide transfer reaction from the ancient charged tRNAs, which were mini RNA helices. This HYPOTHESIS IMPLIES that"Authors' response: Done.(8) In the first paragraph of the subsection entitled "The translational GTPases" it should be noted that Fox & Naik have previously pointed out that molecular mimicry suggests that the EF-Tu-tRNA complex may have preceded EFG.Authors' response: Reference added.(9) In the second paragraph of the subsection entitled "The translational GTPases" the phrase "The IF1 as an OB fold" is unclear- perhaps what is meant is something like "The OB domain found in IF-1 is effectively a small protein etc "Authors' response: As noted below in response to the second reviewer, the entire OB section has been rewritten, and we have also clarified the single OB domain nature of IF1.The manuscript presents a phylogenetic and structural analysis of bacterial and archaeal translation-associated GTPases. The authors point at several regions and domains of bacterial and archaeal GTPases that are structurally similar, while having little similarity in sequence. Using this analysis authors conclude that the earliest event in the evolution of ribosome-associated proteins was the fusion of OB-fold domain with Ras-like GTPase domain. They also suggest that OB-fold mimics ancient tRNA minihelix. This is a very interesting study with results shining light on one of the oldest and most fundamental events in the evolution of life on earth.Results section of the manuscript presents extensive structural analysis that is very hard to follow. My recommendation is to develop a table that summarizes authors' observation on domain-by-domain bases. Numerous three-letter acronyms should also be summarized in a single place in the manuscript.My understanding of the main observations is the following: 1) high sequence and structure diversity of the switch region of all GTPases, (2) high conservation of SRP GTPases and their RNA-binding site, and (3) presence of OB-fold as a second domain of ribosome-associated GTPases. First, I failed to see OB-fold in some proteins, like EF2. In fact SCOP OB-fold domain is absent in all PDB proteins listed in Figure  high seqMy recommendation to the authors to publish the manuscript after introducing changes aimed to (i) clarify and summarize Results section and (ii) establish stronger connections to conclusions.Current Opinion in Structural Biology Volume 12, Issue 6, 2002, pp 794-801) and is found as the second domain of all the ribosomal GTPases in Figure Authors' response: The OB fold is defined in "OB-fold domains: a snapshot of the evolution of sequence, structure and function" by Vickery Arcus (The OB domain of IF1 does interact with the mRNA codon and that of EFTu with the CCA as referenced. See:Trends in Biochem (2003) v. 28, p 434, Gregers R. Andersen, Poul Nissen and Jens Nyborg.Chris Sander: This reviewer provided no comments for publication.


Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified.A search of forty-nine complete fungal genome sequences revealed that, with the exception of Iron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice. All knoAlthough the number of NRPSs encoded by individual filamentous fungi varies from 0 to > 20, most of these and their corresponding metabolites are not conserved across the fungal kingdom, making it difficult to trace the evolutionary history of the corresponding genes. Various evolutionary processes may account for this. The observation that A-T-C modules from a single NRPS often group together as a monophyletic clade suggests tandem duplication of modules as a possible mechanism by which multimodular NRPSs arise . It is cN5-acyl-N5-hydroxy-L-ornithines (AHO) and a ring of three amino acids  strains in terms of sensitivity to ROS, although mutants lacking extracellular siderophores do show increased sensitivity to ROS  and B. cinerea [BC1G10928 and BC1G15494 (Type II)]  and C. cinerea CHGG02251 with the species phylogeny at nodes 9, 10, and 11 where these two NRPSs group with or outside of basidiomycete U. maydis fer3 rather than with other ascomycete NRPSs  and the NPS1/SidC lineages  Fig.  and 6. BA. nidulans SidC are shown in Fig. The unrooted ML phylogenies of A and C domains from The experimental structure of Gramicidin GrsA  bound to3 group toward the carboxyl group of the phenylalanine substrate . ,9. 25,9]AHO and amino acid substrate assignments for A domains are shown in Table Assignment of the remaining A domains was even more difficult. We found that the consensus 10AA codes for SER, ALA, and ORN identified by Stachelhaus et. al.  were notAnSidC has been shown to produce ferricrocin ,48, whicinfer the specificity of the sequences we examined. Instead, A domains predicted to code for the same substrate  had widely divergent 'codes'  and between NPS2 AMP2 and NPS2 AMP4 (θII = .283 ± .113) were significant at p ≤ .05. Several positions received high support from all three methods including positions 252, 278, 301, 322, and 331. Several of the positions identified by structural modeling  also received support from at least one method  or a shift in evolutionary rate between clusters reflective of changes in evolutionary constraint or selective pressure (Type I) Table . For Typod Table . Two excC. heterostrophus NPS2 and A. nidulans SidC. Some fungi contain representatives of both lineages while others lack a ferrichrome synthetase altogether. Significantly, ferrichrome NRPSs were not detected in any yeast species sampled , or in the zygomycetes R. oryzae and P. blakesleeanus, the ectomycorrhizal fungus L. bicolor or the chytrid B. dendrobatitis. While absence of a gene must be interpreted with caution, as genome sequences may be incomplete, the lack of the NPS1/SidC lineage in all Dothideomycetes  and Onygenales , lack of the NPS2 lineage in Eurotiales (Aspergillus sp.), as well as a lack of any ferrichrome synthetase in all hemiascomycete yeasts, zygomycetes, or chytrids surveyed is likely significant.Our phylogenetic analyses support the hypothesis that fungal ferrichrome synthetases fall into two distinct lineages corresponding to homologs of U. maydis (Umfer3 and Umsid2) and the ascomycete B. cinerea (BC1G10928 and BC1G15494). This scenario would postulate an unlikely loss of one or the other of these genes in the majority of species examined. The other possibility is independent duplication of the NPS1/SidC type gene in certain species e.g., U. maydis and B. cinerea. However, in both ML and Bayesian phylogenetic analyses, the ascomycete proteins B. cinerea BC1G15494 and F. graminearum FG11026 grouped with, or outside of, basidiomycete proteins, suggesting an ancestral duplication of this lineage , has recently been shown to produce malonichrome   while certain other ascomycete members  of the NPS1/SidC lineage produce ferricrocin . The two ferrichrome synthetases in U. maydis also produce distinct products; Umfer3 produces ferrichrome A  and Umsid2 produces ferrichrome (3 GLY).It is possible that the duplications within the NPS1/SidC lineage may be associated with production of different ferrichromes. C. heterostrophus NPS2 C domains  provides support for the notion of an ancestral gene with six complete modular units. Furthermore, our analyses (unpublished) and others [These interpretations are based on algorithms for which it is assumed that there is no loss and no recombination, criteria that are clearly violated here for ferrichrome synthetases. We propose, however, that the C domains of d others  show litC. heterostrophus (representative of Type V) and F. graminearum (representative of Type IV) have 6 C domains, although they have only 4 and 3 A domains, respectively. Analyses of C domains of these proteins clearly indicate that the second C domain of Types V and IV are related . Similarly, the second C domain in sib1 from S. pombe groups with the second C domain in C. heterostrophus NPS2 but lacks the corresponding A domain  and the degenerate second A domain  which consists of three AHO residues only.Type VI The mechanisms controlling iterative use of NRPS domains are, to our knowledge, unknown. Here we observe that proteins with distinct domain architectures produce nearly identical chemical products. Iterative synthesis provides yet another flexible mechanism for NRPS biosynthesis.Structural modeling results suggest that general features of the binding pocket such as size, hydrophobicity, and charge may be more important in determining substrate recognition than residues at fixed positions within the cavity. In homology based modeling of substrate specificity, small errors in the alignment between the experimental and the model sequence can lead to significant errors in the modeled structure. For this reason, we used an alignment of several experimental structures to optimize our alignments. We found that the A domains included in this study were remarkably conserved structurally and we were able to identify several conserved residue-patterns and structural features which aligned well in all the structures and served as markers to anchor our alignment of the experimental sequences, particularly near the residues that are supposed to form the wall of the binding site (the code). With careful attention to the alignment, we found that residues associated with the 10 or 13 AA 'codes' predicted to be important in substrate choice vary considerably and do not show a consistent pattern for A domains predicted to code the same substrate Table . Thus, wOur results from structural modeling and evolutionary analyses of functional residues point to key fragments within the binding pocket which surround and connect the α-helix and β-strand structural elements of the pocket, as general regions important for specificity. Our analyses also identified residue positions in addition to the 10 AA code positions within these fragments  which line the substrate pocket and are either positioned such that their side chains may interact with a substrate or are involved in shaping the size of the binding pocket  could have a role in either protein-protein interactions or interactions between the two subunits of the NRPS protein. One subunit contains both the substrate and AMP binding pockets while the other subunit covers the opening to the binding sites Fig. . In the Thus, our results suggest that a rigid 'code' of specific amino acids at particular residue positions may not be the most reliable approach to predicting specificity of fungal NRPS A domains. Instead, the general chemical, physical, and structural features of the binding pocket may be more important. We conclude that methods of substrate prediction which evaluate chemical features of amino acids within these key regions may be better able to predict substrate specificity. Our findings await manipulation of key residues predicted to affect the chemical properties of the binding pocket, followed by examination of how this affects substrate choice.Our results demonstrate two distinct lineages of ferrichrome synthetases in fungi and suggest that these genes are restricted to fission yeast, filamentous ascomycetes, and basidiomycetes. Phylogenetic analyses of domain architectures supports the hypothesis that the distinct domain architectures observed derive from a hexamodular ancestral gene through loss of individual A domains or complete A-T-C modules and support a series of tandem duplication events of single or double A-T-C modules as the mechanism generating this hexamodular ancestor.Analyses of substrate specificity show that the proposed 10AA code was unable to infer substrate specificity for these fungal A domains. Structural modeling and evolutionary analyses of functional residues suggest that additional positions may play a role in substrate specificity. Our results confirm that two positions of the code which are conserved across all sequences examined, D235 and K517, likely do not play a role in amino acid substrate choice but instead serve the important function of anchoring the substrate in the binding pocket through interaction with the amino and carboxyl groups respectively.KEB selected and performed most of the phylogenetic analyses. DRR performed the protein structural modeling. BGT directed the research. KEB and BGT wrote the manuscript, with input from DRR.Protein accession numbers used in this study. GenBank accession numbers of proteins in this study cross referenced to genome IDs where available.Click here for fileSpecies tree. Species tree used for reconciliation analyses (See additional file Click here for fileBayesian analyses of all AMP domains examined in this study. Alternative phylogenetic method to the maximum likelihood analysis of A domains from the complete dataset, provided for comparison (See additional file Click here for fileIndividual NPS2 and NPS1/SidC A and C domain lineage analyses. The data provided represent separate phylogenetic analyses of NPS2 and NPS1/SidC adenylation and condensation domains (See additional file Click here for fileAmino acids corresponding to the 10AA code positions of selected bacterial and fungal NRPS adenylation domains. These data show the 10AA code for selected bacterial and fungal NRPS A domains described in the literature, or reported in this work, as coding for glycine, alanine, serine, or ornithine.Click here for fileAdditional file4figure legends. Figure legends for additional files Click here for file


They often possess exceptionally long N-terminal signal sequences. We analyzed 90 long signal sequences of bacterial autotransporters and members of the two-partner secretion pathway in silico and describe common domain organization found in 79 of these sequences. The domains are in agreement with previously published experimental data. Our algorithmic approach allows for the systematic identification of functionally different domains in long signal sequences.Bacterial autotransporters represent a diverse family of proteins that autonomously translocate across the inner membrane of Gram-negative bacteria  The SwissProt database entries contain sequences with predicted or putative signal sequences.We analyzed 16 autotransporters and two-partner secretion sequences published by Szabady et alThe following sequences were omitted from our analysis due to minor sequence aberrations between the publications and the UniProtKB database entry: O32591, Q47692, Q54151 and Q8VSL2.The following to YP_001161762 orthologous sequences were omitted since they possess an identical signal sequence: YP_001719317, YP_001874066, Q1C309, Q1CMJ2 and Q665P5. When two database entries contained the identical sequences one entry was omitted and both accession numbers are given.In total, 90 signal sequences encompassing more than 40 residues from bacterial autotransporters were analyzed in this study and in regards to their possible internal domain organization.The 28 long signal sequences not associated with autotransporters were retrivied from the UniProtKB/SwissProt Release 14.7The 228 short bacterial signal peptides associated with autotransporters were retrieved from the UniProtKB/SwissProt Release 14.7in vitro, from which five exhibit the predicted targeting functionThe detection of the domains was performed using the NtraC algorithm,We analyzed 90 long signal sequences of bacterial autotransporters and the two-partner secretion pathway in regards to their potential two-domain (NtraC) organization.Of the 16 signal sequences collected in Szabady et alIn total, from 90 long signal sequences considered in this study 77 (86%) are predicted, by our algorithm, to be organized in two domains.For two additional sequences  an NtraC organization is predicted which in the context of this work could be regarded as a false-positive: No C-domain with a targeting capacity was detected. For Q2J0N4 an N-terminal mTP is predicted by TargetPThe two-domain organization proposed by the algorithm is in agreement with the ESPR of Desvaux et alvia SRS, the conserved pattern is only present in three sequences : unknown function,– C-domain (residues 27–55): predicted secretion signal for Gram-negative bacteria.The algorithm thereby proposed the same functional domain Peterson et al described experimentally.We would like to stress that the NtraC algorithm is based on sequence information only and not influenced by the existing proposed fragmentation of long signal peptides. Our prediction method is therefore unbiased for the analysis of new sequences.A further surprising result is the prediction of mitochondrial targeting peptides (mTP) for the proposed N-domains of the long bacterial signal peptides. In 17 of 90 (19%) cases the N-domain of a bacterial signal sequence is predicted as mTP . Short bThe automatic assignment “mTP” should thus not to be regarded as a perfect functional prediction but as the detection of a feature, namely the high abundance of charged residues. In 1994 Izard and Kendall reported that although a positive charge in the N-terminus may not be absolutely required for secretion20in silico in the context of long signal sequences of autotransporters and their potential domain organization we counted the occurrence of charged residues in the N- and C-domain of all 79 autotransporter sequences predicted to be two-domain organized (tra) or excluded (−tra) from the domains for the calculation (To investigate the role of charged residues rganized . The borculation .tra), positively charged residues  occur approximately 1.6 times more often in the N-domain compared to the C-domain. Negatively charged residues  occur 2.3 times more often in the N-domain(−tra) compared to the C-domain(+tra). This difference becomes even more prominent if the border between both domains is counted as part of the N-domain(+tra) leading to 2.8 times higher occurrence of positively charged residues and 4.2 times higher occurrence of negatively charged residues in the N-domain compared to the C-domain.If the border between the domains was regarded as part of the C-domain(+tra) and C-domain(−tra) indicates that not only the presence of charged residues is of importance but also their position, favoring the N-terminal domain or between the two domains. The relative position curtly before the targeting signal in the C-domain could represent a characteristic feature.This charge bias is an argument that charged residues may represent an inherent difference between the N- and the C-domain. The nearly three-fold increase in deviance between the N- long signal peptide sequences of long autransporters and 21 out of 28 (75%) long bacterial signal peptides not associated with autotransporters could form an amphipathic helix. We compared this to short bacterial signal peptides associated with autotransporters and found that 34 out of 228 (15%) could form an amphipathic helix. The requirement to form an amphipathic helix was to possess nine adjacent amino acids in a helix in a window of 18 residues. In a second approach we allowed the adjacent nine e.g. polar residues to be interrupted by one e.g. non-polar residue and vice versa. Now 68 out of 90 (76%), 25 out of 28 (89%) and 58 out of 228 (25%) could form an amphipathic helix (A further hint towards a mechanistic aspect arises from to the secondary structure aspect of the NtraC model. As the C-domain of the signal peptide with its hydrophobic core is embedded in the membrane or the Sec complex during translocation, the N-domain may be kept in a defined angle to the membrane due to a predicted β-turn in the border between the N- and C-domain. The positive net charged of the N-domain could have the effect of keeping it outside and on top of the membrane. This might provide the means for the recruitment of other proteins . We furtic helix . While oin silico in long signal sequences. We corroborate the importance of charged residues in bacterial signal sequences and emphasize their position near the N-terminus as possible regularity. The approach highlights the relevance of charged residues in long signal sequences.We present an extensive analysis of 90 long bacterial autotransporter signal sequences predicting in 86% of the sequences, a common two-domain organization. The described organization is in agreement with published experimental data and allows the identification of potential new domains Table S128 long signal sequences not associated with autotransporters.Table S2228 short bacterial signal peptides associated with autransporters.


Homo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster. On the basis oftheir domain combination and function, PKLNKs have been classified mainly into fourcategories: (1) Ligand binding PKLNKs, (2) PKLNKs with extracellular protein-proteininteraction domain, (3) PKLNKs involved in dimerization, and (4) PKLNKs with cytoplasmicprotein-protein interaction module. While members of the first two classes of PKLNKshave transmembrane domain tethered to the PKLNK domain, members of the other twoclasses of PKLNKs are cytoplasmic in nature. The current classification scheme hopes toprovide a convenient framework to classify the PKLNKs from other eukaryotes whichwould be helpful in deciphering their roles in cellular processes.Protein Kinase-Like Non-kinases (PKLNKs), which are closely related to protein kinases,lack the crucial catalytic aspartate in the catalytic loop, and hence cannot function asprotein kinase, have been analysed. Using various sensitive sequence analysis methods, wehave recognized 82 PKLNKs from four higher eukaryotic organisms, namely,  It is now well known that enzymes, in their role as biocatalysts, are the most important control points in the living organisms, and the catalytic residues of an enzyme are key to its molecular function. Bartlett and colleagues  have desβ-turn structure with 2 or 3 glycine residues (G-X-G-X-X-G) while subdomain II comprises an invariant lysine participating in anchoring and orienting the ATP (Adenosine Tri Phosphate). Subdomain VIB contains catalytic loop with a key aspartate [D] residue that mediates the transfer of a phosphate group from ATP to the appropriate substrate. The D residue of DFG motif in subdomain VII ligates Mg2+ which in turn interacts with β and γ phosphates of ATP [Phosphorylation by Ser/Thr/Tyr protein kinases plays a crucial role in cellular signal transduction. A canonical kinase domain consists of 12 subdomains containing few conserved residues of functional importance. Subdomains I, II, VIB, and VII are considered to be the most important ones. Subdomain I includes Although the importance of protein kinases has long been recognized, studies on protein kinase homologues lacking catalytic residue/residues are more recent. Several studies on repertoire of kinases in various organisms have revealed presence of enzymatically inactive homologues of protein kinases –6 which Despite considerable sequence similarity to enzymatically active protein kinases, Protein Kinase-Like Nonkinase  domains lacking key residues thought to have regulatory roles. Some examples of proteins containing such domains which lack catalytic base aspartate are a PKLNK domain tethered to a tyrosine kinase domain in Janus Kinase (JAK) and membrane guanylyl cyclases (or particulate guanylyl cyclase) in which a regulatory PKLNK domain is situated N-terminal to the guanylyl cyclase domain –10. PKLNIn the past, our group has reported presence of ABC1, RIO1, and kinases in archaea and bacteria that share significant similarity with Ser/Thr/Tyr kinase family . The seqHomo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster. An attempt has been made to classify these PKLNKs based upon their amino acid sequences and domain tethering preference in order to understand molecular basis of evolution and functions of these proteins.In the current analysis, we present a detailed analysis of the PKLNKs from four completely sequenced higher eukaryotes, namely, In order to identify the repertoire of PKLNKs in various eukaryotic organisms PSI-BLAST  search wThe subfamily classification of these PKLNKs and recognition of other domains in the PKLNK domain containing multidomain proteins have been made using the procedures and protocols developed earlier in our group in connection with analysis of kinomes , 6, 21. In the current analysis, we have identified a total 82 PKLNKs in the four organisms. CD-hits program , 27 was CLUSTALW  has beenhttp://pfam.sanger.ac.uk/). MulPSSM (Multiple PSSM) [Domain assignment to the other regions apart from the noncatalytic kinase domain of these PKLNKs has been carried out using HMMer search methods by querying each of the PKLNK against the 10340 protein families HMMs available in the Pfam database (le PSSM)  approachle PSSM) .Homo sapiens . There are 31 PKLNKs identified in  sapiens , 18 PKLNnogaster , 13 PKLNrvegicus , and 20 musculus . AlthougThough these PKLNKs lack the crucial aspartate in the catalytic loop, they are closely related to the functional protein kinases, in terms of the sequence similarity.In the multidomain proteins a given domain acts in conjunction with other domains which are tethered together in the same polypeptide chain and help in their regulation. Absence of catalytic aspartate in the active site reflects the noncatalytic activity of PKLNKs, but domain organization of a protein can give clues about putative functional roles. Domains tethered to PKLNKs suggest that though these PKLNKs lack key catalytic residues, they are involved in protein-protein interactions and might have important regulatory roles in signal transduction pathway. In the current analysis we have identified 19 different types of domains tethered to the PKLNK domain. Except 24 PKLNKs, majority of PKLNKs identified have accessory domains tethered to them . The PfaBased upon the broad function, the domains tethered to PKLNK can be functionally categorized into four categories:Domains which are mainly involved in ligand binding like ANF receptor, Receptor L domain, and Ephrin receptor ligand binding domain. There are 17 gene products which have domains architecture similar to ANP receptor  in which(2)Domains which are extracellular and involved in protein-protein interactions like I-set (Immunoglobulin like domain) and Fn3 (Fibronectin type III) domains.(3)Domains involved in dimerization like Death domain, SAM (Sterile Alpha Motif) domain, and Furin-like domain.Proteins containing death domains are well known to participate in the signaling events which regulate apoptosis  indicati(4)Domains involved in protein-protein interactions like Ank (Ankyrin repeats) and Heat repeats. Ank is one of the most common protein-protein interaction modules which occur in large number of functionally diverse proteins. PKLNKs containing Ank repeat are likely to play role in diverse functions like signal transduction, ion transportation, transcription initiation, and so forth. Heat domain is 30–40 amino acid tandemly repeated domain. PKLNK containing Heat domain might have role in intracellular transport processes.Apart from the domains discussed above there are some more accessory domains found tethered to the PKLNK domain which provide functional diversity to the PKLNKs. A human PKLNK (gi|18676872) has TBC domain and Rhodanese domain in the C-terminal. TBC domain is involved in GTPase signaling, and Rhodanese domain which shares evolutionary relationship with large family of protein is involved in cyanide detoxification . AnotherA drosophila PKLNK (gi|17368346) has WSC domains N-terminal to the PKLNK domain which is likely to be an extracellular carbohydrate binding domains. At least three PKLNKs  have PX domain N-terminal to the PKLNK domain which might have role in lipid signaling.Phylogenetic tree has been generated by considering the nonenzymatic PKLNK domains of these 82 PKLNKs  in whichThe biological function of these PKLNKs might be in the regulation of tyrosine protein kinase activity.Further, we have compared the domain structure of PKLNKs and their closest protein kinase subfamilies. Interestingly, we have observed that there are a few domain combinations which are unique to either PKLNKs or protein kinases. There are a few Pfam domains such as TUDOR and HNOBA (Heme NO binding associated) which have not been seen tethered to protein kinase domains so far. HNOBA domain is known to function as heme-dependent sensor for gaseous ligands and transduce diverse downstream signals across diverse organisms . The domhttp://www.hprd.org/) [Understanding the biological roles of proteins in the cellular environment is the main aim of genome analysis. For almost all cellular processes in a living cell protein-protein interactions are of central importance. In the current section, we have focused on human PKLNKs. We have looked for the protein-protein interactions of PKLNKs using HPRD database (rd.org/) . At leasHomo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster strongly argues against pseudogenes, as otherwise these would have been lost during the evolutionary time. Our study on PKLNKs suggests that most noncatalytic PKLNKs are derived from active protein kinase ancestors and have lost one or more of the critical catalytic residues within the active site which provides new insight into nature's way of eliciting new functions of PKLNKs. Based upon the domain tethering preferences we have classified PKLNKs into four main classes in which members of the two classes are receptor PKLNKs which are mainly involved in ligand binding and protein-protein interaction extracellularly while other two classes of PKLNKs have members which are cytoplasmic, and they are mainly involved in dimerization and protein-protein interaction in the cytoplasm. The phylogenetic analysis reveals function-based clustering of these PKLNKs. Conservation of some of the modular organization across the four organisms suggests their central role in the eukaryotic signaling pathway. Since many of the PKLNKs have other domains tethered to them and are involved in protein-protein interactions, one can speculate that though the kinase-like domain is nonenzymatic, they might have role in regulation and scaffolding. Some of these catalytically inactive members of PKLNKs which are close homologues of the receptor tyrosine kinase are shown to be over-expressed in cancer cells. Additional studies are required to determine precise function and role of these PKLNKs in tumorgenesis and its usefulness in the diagnosis of tumors. Domain organization of these PKLNKs revealed that some of the PKLNKs have new and hence unique domain organization so far not seen in any other family of gene products. 3D structure and biochemical analysis can further determine and explore the functional role of these PKLNKs. The presence of putative PKLNK in higher eukaryotes indicates that we have more to learn about cellular signaling involving these noncatalytic domains. Evolutionary history of these PKLNKs would be of particular interest. It is hoped that this analysis will provide a better understanding about the frequent occurrence of PKLNKs in different organisms and hence their function.This work represents functional analysis of noncatalytic PKLNKs across a data set of 82 PKLNKs from four higher eukaryotes. Our analysis has indicated that existence of noncatalytic PKLNKs is quite common. The fact that noncatalytic PKLNKs are well conserved between Multiple sequence alignment of the PKLNK domains of human, drosophila, mouse and rat. The site of replacement of catalytic residue is highlighted in red.Click here for additional data file.


The general method used to determine the function of newly discovered proteins is to transfer annotations from well-characterized homologous proteins. The process of selecting homologous proteins can largely be classified into sequence-based and domain-based approaches. Domain-based methods have several advantages for identifying distant homology and homology among proteins with multiple domains, as compared to sequence-based methods. However, these methods are challenged by large families defined by 'promiscuous' (or 'mobile') domains.Here we present a measure, called Weighed Domain Architecture Comparison (WDAC), of domain architecture similarity, which can be used to identify homolog of multidomain proteins. To distinguish these promiscuous domains from conventional protein domains, we assigned a weight score to Pfam domain extracted from RefSeq proteins, based on its abundance and versatility. To measure the similarity of two domain architectures, cosine similarity  is used. We combined sequence similarity with domain architecture comparisons to identify proteins belonging to the same domain architecture. Using human and nematode proteomes, we compared WDAC with an unweighted domain architecture method (DAC) to evaluate the effectiveness of domain weight scores. We found that WDAC is better at identifying homology among multidomain proteins.Our analysis indicates that considering domain weight scores in domain architecture comparisons improves protein homology identification. We developed a web-based server to allow users to compare their proteins with protein domain architectures. Homology identification is part of a broad spectrum of genomic analyses, including the annotation of new whole genome sequences, the construction of comparative maps, the analysis of whole genome duplications and comparative approaches to identifying regulatory motifs . The genDomain-based methods use information of the domains contained in proteins . DomainsDomain-based approaches identify homologous proteins generally by comparing protein domain architecture, which is the linear order of the individual domains in multidomain protein. About two thirds of proteins in prokaryotes and 80% of proteins in eukaryotes are multi-domain proteins . Studieshttp://wdac.kr/.Here we present a measure, called Weighed Domain Architecture Comparison (WDAC), of domain architecture similarity, which can be used to identify homologs of multidomain proteins. The key ideas are the use of weight scores for domain promiscuity and combining domain architecture comparison with sequence similarity method. The weight scores are calculated based on a domain's frequency and versatility in RefSeq  proteinsIn this study we used the Pfam  databased, isTo measure the strength of domain promiscuity, we considered two features of protein domains, the first of which is domain abundance. Compared to non-promiscuous domains, promiscuous domains appear in many proteins because they are needed to perform auxiliary functions. Vogel et al.  have shopt is the number of total proteins and pd is the number of proteins containing domain d.where d, isThe second feature of protein domains that we consider is domain versatility. Promiscuous domains occurring in many protein clusters have many partner domain families while highly conserved domains appear in a small number of protein clusters and their neighbor domains are also conserved during evolution . Thus, dfd is the number of distinct domain families adjacent to domain d. The weight score of a domain is simply calculated by the product of the IDF and the IV of a domain. Let us consider the theoretical example  . Domain x and y have the same domains and 0 indicates that they share no domains.The range of the cosine similarity is , where 1 indicates that Qs) by the total domain pairs (Qt). The function is defined bySecond, domain orders were considered. To measure the order similarity, we compared shared domain pairs between two domain architectures. In domain evolution, two- or three-domain combinations, called supradomain, are re-used in different protein context, and domain pairs in protein domain architecture occur in only one order, with only about 2% of such pairs occurring on both possible orders. The order similarity is measured by dividing the shared domain pairs  using a simple linear function.The final similarity score between two domain architectures, We constructed an automatic pipeline for identifying homologs of proteins Figure . The pipWe developed a web-based server to provide a back-end pipeline for protein homology and to allow users to compare their protein sequences with a domain architecture database. The web interface is implemented with static HTML and CGI scripts, and MySQL DBMS is used to store the database.We downloaded 6,042,750 protein sequences from the RefSeq database (Release 32). The domain content of the sequences was analyzed with Pfam 23.0 containing 10,340 families. The Pfam domain annotations of all RefSeq proteins were obtained from the Similarity Matrix of Proteins (SIMAP)  databaseOf the 6,042,750 RefSeq proteins, 3,942,678 (65%) contain more than one Pfam domain. These Pfam-annotated proteins were converted into domain architectures, in which we obtained 55,841 distinct domain architectures. The domain architecture data show that 90% of the domain architectures are kingdom-specific. Thus, we classified the 55,841 domain architectures into three kingdoms: Eukaryote, Bacteria, and Archaea , appearing in 54,980 bacterial proteins. To measure domain versatility, we obtained kingdom-specific N- and C-side distinct domain families adjacent to each domain. We found that most domains have one or two distinct adjacent domain families. The features of the obtained domain versatility are consistent with earlier reports that the number of different partner domains for a single domain or for a domain combination follows a power law distribution: many domains or domain combinations have only very few different N-terminal or C-terminal partner domains. The most versatile domain in the three kingdoms is Ank (PF00023), having 220 distinct partner domain families in eukaryotes.We calculated kingdom-specific IDF and IV scores for all domains using eq. 1 and 2, and obtained weight scores for each domain by the product of the IAF and IV scores. The scores were multiplied by 10 to facilitate computation. These domain's scores represent their importance in the protein universe and are used in the comparison of domain architectures. The analysis of the weight scores indicates that they are distributed 0.2 to 138.00  and Caenorhabditis elegans (nematode) protein sequences. In the DAC method, domain weight scores are not considered. To implement the DAC method, we used Jaccard similarity [To assess the effect of domain weight scores on domain architecture comparison, the WDAC (weighted method) was compared to the general unweighted domain architecture comparison (DAC) method using all complete milarity , which if11 is the number of domains common to both sequences X and Y, f10 is the number of domains in X, and f01 is the number of domains in Y.where We extracted all complete human and nematode protein sequences from RefSeq proteins, yielding 32,999 human and 23,220 nematode protein sequences. Among these proteins, 23,295 human and 14,522 nematode proteins have detectable Pfam domain information. Among the human proteins, we selected 9,764 proteins that contain more than 2 Pfam domains and performed domain architecture comparisons between the selected human proteins (≥ two domains) and those from the nematode proteome (≥ one domain) using the WDAC and DAC algorithms.To validate homologous pairs of human and nematode proteins, we used the HomoloGene database , a NCBI In addition, we found that the WDAC results have more specific homologs than the DAC results. Figure The query interface accepts protein sequences in FASTA format, and the maximum number of input protein sequences for a single submission is 100 and the length of each sequence is limited to 5000 residues. The output of the server is an HTML-formatted file, which consists of three parts: query domain architecture with Pfam domains, matched domain architecture, and domain information Figure . For morThere are several current homology methods which compare domain architectures. However, these methods are challenged by large families defined by promiscuous domains. To cope with the promiscuous domain problem, we present a method for measuring the similarity among protein domain architectures based on their Pfam-A domain annotations. The Pfam database may contain a small number of false positives and false negatives. Nevertheless, it is currently one of the most useful and practical domain annotation databases for protein sequences. In this study, we consider domain weight scores, obtained based on the abundance and versatility of domains. Our analysis indicates that considering domain weight scores in domain architecture comparison improves the performance of protein homology identification. The WDAC algorithm is also effective in resolving some issues that have baffled traditional sequence-based comparison methods, such as the comparison of proteins with promiscuous domain(s). The WDAC algorithm and its web server could be used to explore the underlying evolutionary relationships among proteins at the level of their whole domain architectures, rather than at the single-domain or protein sequence level.The authors declare that they have no competing interests.BL designed the algorithm, carried out the programming, analyzed the results and wrote the manuscript. DL directed the entire study. All authors read and approved the final manuscript.BMC Genomics Volume 10 Supplement 3, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Computational Biology, available online at http://www.biomedcentral.com/1471-2164/10?issue=S3.Other papers from the meeting have been published as part of


The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors.Arabidopsis Toc159 family members (atToc132 and atToc159) are disordered at physiological pH and temperature and undergo conformational changes at temperature and pH extremes that are characteristic of IDPs.The physicochemical properties of the A-domains are characteristic of intrinsically disordered proteins (IDPs). Using CD spectroscopy we show that the A-domains of two Identification of the A-domains as IDPs will be important for determining their precise function(s), and suggests a role in protein-protein interactions, which may explain how these proteins serve as receptors for such a wide variety of preprotein substrates. Arabidopsis, two families of GTPases are responsible for preprotein recognition; the Toc34 and Toc159 receptors [Arabidopsis: atToc159, -132, -120 and -90 [Arabidopsis family members share ~65% sequence identity [Arabidopsis (~20% identity) and has no known conserved functional motifs [Most chloroplast proteins are encoded in the nucleus and translated in the cytosol with an N-terminal transit peptide that facilitates recognition by receptors of the Toc complex. In eceptors . Toc159 eceptors  at earlyeceptors ,7, sugge and -90 ,9. These and -90 ,8,10-12.identity -16, compidentity . Less inl motifs ,18, the l motifs . DespiteArabidopsis proteins [Based on hydrophobic cluster analysis of its A-domain, Toc159 has been proposed to belong to a growing class of natively unstructured or intrinsically disordered proteins (IDPs) , which sproteins .Arabidopsis Toc159 family, atToc159 and atToc132, are IDPs. This represents the first investigation into the structure of the A-domains of the Toc159 family, and has implications for future studies aimed at understanding the function of these domains, and the role of Toc159 receptors in general, in chloroplast protein import.IDP domains are involved in highly dynamic protein-protein interactions ,22, oftePisum sativum (psToc159) (Accession AAF75761) are also predicted to be largely disordered (data not shown). A-domains of atToc159 and atToc132 were selected as representatives for further examination.A recent study led to the suggestion that the A-domain of atToc159 may be natively unfolded . In the E. coli-expressed A-domains of atToc132 and atToc159 possessing N-terminal His6 tags (132AHis and 159AHis) were purified using Ni2+-charged resin  to 49% (at 65°C), and again, there is a concomitant gain in β-sheet content from 22% to 43%, and in α-helical content from 3% to 7%  and 4.0 (159AHis). Presumably, a decrease in net charge leads to a decrease in electrostatic repulsion between negatively charged residues, allowing for partial folding. An increase in structure for 132AHis at low pH can also be detected using fluorescence spectroscopy  that fluoresce when excited at 295 nm . The fluorescence maximum of 132AHis shifts to a lower wavelength at pH 3, suggesting that the Trp residues are less exposed to solvent as a result of partial folding at low pH - a commonly observed phenomenon of acidic IDPs [To further characterize the structural properties of the A-domains, the effects of temperature and pH on the conformation of 132Ae Figure . Spectra7% Table . Specifiy Figure , inset. dic IDPs ,32.His and 159AHis show a notable increase in secondary structure from ~3-5% α-helix, 22-32% β-sheet in the absence of TFE to 28% α-helix, 28% β-sheet in 50% TFE  both 132AE Figure , Table 1His gained more structure than 159AHis under these conditions. These differences in conformational flexibility could reflect functional differences between the A-domains of atToc132 and atToc159.Interestingly, the A-domains showed differences in the amount and types of secondary structure gained at low pH and in the presence of 50% TFE. In particular, 132AAs part of an evolutionary study into the origin of Toc159, it was suggested that the A-domain of atToc159 might be natively unfolded . In the His and 132AHis in this study may be reflective of such an ability to discriminate between preproteins. In addition, it has been proposed that IDPs with large surface area may act in a "fly-casting" mechanism to increase the speed of low affinity protein-protein interactions [in vitro [Finally, perhaps the most intriguing potential function for the A-domain that emerges from the finding that it is an IDP is a role in transit peptide recognition. Transit peptides are variable in length  and sequence, are rich in hydroxylated amino acids, scarce in acidic amino acids, and lack a defined three-dimensional structure in aqueous solution -39. It iractions , which win vitro , as wellin vitro . Analogoin vitro ,47.While interactions between the Toc159 family A-domains and transit peptides is an attractive mechanism for transit peptide recognition, detection of transient protein-protein interactions is technically challenging, and it is not yet clear whether the techniques employed here will be sufficient to detect potential interactions between the A-domain and transit peptides. CD may prove useful in this regard if association is accompanied by a disorder-to-order transition ; the acqIn summary, the A-domain represents a large portion of the Toc159 receptors and differs significantly among members of this family. The function(s) of this domain, however, has remained elusive. In this study, the structure of the A-domains has been investigated for the first time. The finding that the A-domains are intrinsically disordered has implications for understanding their function(s), and future studies on the Toc159 receptors will be aimed at identifying A-domain binding partners to help elucidate the role of this domain in chloroplast protein import.atTOC132 and atTOC159 cDNAs, which correspond to the A-domains of atToc132 (132A) and atToc159 (159A), respectively, were sub-cloned by PCR using cDNA clones as templates [6-tag, and 5'NheI/3'SacI (132A) or 5'NheI/3'SalI (159A) restriction sites for sub-cloning into pET21b (Novagen). Final constructs encode recombinant atToc132A and atToc159A including N-terminal hexahistidine tags and are denoted 132AHis and 159AHis. The proteins were overexpressed in E. coli BL21(DE3) using standard conditions. Cells were lysed in lysis buffer  using a French press. Total soluble protein was applied to a Ni2+-NTA column (Novagen) at 4°C. The resin was washed with lysis buffer containing 30 mM imidazole, and proteins were eluted with lysis buffer containing 250 mM imidazole. 132AHis and 159AHis were further purified using a batch method of ion exchange. Briefly, Ni2+-purified protein was diluted 1:1 in ion exchange buffer  and incubated with 1.5 mL of a strong-anion exchange resin  for 10 min at room temperature on a rotating mixer. Protein was eluted with a 20 mM piperazine solution at pH 4.5, containing 550 mM NaCl. The purified protein was concentrated and exchanged into CD Buffer  using centrifugal diafiltration . Recombinant 132AHis and 159AHis were analyzed by SDS-PAGE (10% resolving gels) stained with Coomassie, or probed on Western blots using antibodies against the A-domain of atToc132 or atToc159 (see below). Protein concentration was determined using the Bradford assay [The first 1365 or 2181 basepairs of the emplates ,15. PrimHis (~1 μg) and 159AHis (~10 ng) were transferred to nitrocellulose by first soaking the gel in transfer buffer  SDS, and 10% (w/v) methanol). Transfer to nitrocellulose was achieved using a Semi-Dry transfer cell (Bio-Rad Laboratories Inc.) at 15 volts for 90 min. The nitrocellulose was stained with amido black  to confirm successful transfer, and the membrane was blocked by incubating with 5% (w/v) powdered milk in TBS containing 0.1% Tween-20  Tween-20). The membrane was washed with TBS-T and incubated with primary antibody at room temperature for 2 h. Primary antibodies used were rabbit antibodies raised against the A-domain of atToc159 (α-159A) diluted 1:2000, or the A-domain of atToc132 (α-132A) diluted 1:5000 [SDS-PAGE-resolved 132A159AHis ~ ng were His and 159AHis were measured at concentrations of 5 μM or 2.5 μM in CD buffer. Samples were equilibrated at the indicated temperature for 10 min prior to measurements, and pH was adjusted immediately prior to measurement for pH-dependent experiments. Spectra of protein samples and the buffer baseline were measured with a 0.5 nm/s scanning speed at 0.5 nm intervals, and were an average of four scans. Averaged buffer baseline spectra were subtracted from averaged protein sample spectra and the resultant corrected spectra were converted to mean residue ellipticity. Spectra were deconvoluted on the Dichroweb website [Far-UV CD spectra were measured on an Aviv 215 spectropolarimeter . Measurements were performed using rectangular quartz cells with 0.1 cm pathlength. 132A website  using th website .LGLR participated in planning the study, carried out all the experiments and associated analysis, and drafted the original manuscript. MJ-N participated in the planning and interpretation of the biophysical experiments, and contributed to the preparation of the manuscript. MDS conceived of the study, participated in its design, oversaw the experiments, and helped to draft the manuscript. All authors read and approved of the final manuscript.


Polycomb group (PcG) proteins are a set of chromatin-modifying proteins that play a key role in epigenetic gene regulation. The PcG proteins form large multiprotein complexes with different activities. The two best-characterized PcG complexes are the PcG repressive complex 1 (PRC1) and 2 (PRC2) that respectively possess histone 2A lysine 119 E3 ubiquitin ligase and histone 3 lysine 27 methyltransferase activities. While PRC2-like complexes are conserved throughout the eukaryotic kingdoms, PRC1-like complexes have only been described in Drosophila and vertebrates. Since both complexes are required for the gene silencing mechanism in Drosophila and vertebrates, how PRC1 function is realized in organisms that apparently lack PRC1 such as plants, is so far unknown. In vertebrates, PRC1 includes three proteins, Ring1B, Ring1A, and Bmi-1 that form an E3 ubiquitin ligase complex. These PRC1 proteins have an N-terminally located Ring finger domain associated to a poorly characterized conserved C-terminal region.We obtained statistically significant evidences of sequence similarity between the C-terminal region of the PRC1 Ring finger proteins and the ubiquitin (Ubq)-like family proteins, thus defining a new Ubq-like domain, the RAWUL domain. In addition, our analysis revealed the existence of plant and worm proteins that display the conserved combination of a Ring finger domain at the N-terminus and a RAWUL domain at the C-terminus.Analysis of the conserved domain architecture among PRC1 Ring finger proteins revealed the existence of long sought PRC1 protein orthologs in these organisms, suggesting the functional conservation of PRC1 throughout higher eukaryotes. Polycomb group (PcG) proteins are epigenetic gene regulators implicated in important cellular and developmental processes. In animals, PcG proteins were primarily known for their role in maintaining cell identity during the establishment of the body plan , but recCaenorhabditis elegans . Ho. Ho54]. 0%, the hThe most characteristic features of this domain is the α2 that is a bit larger in its C-terminus compared to classical Ubq-like proteins and contains a conserved pair of Thr/Leu that might have a structural role in capping the alpha helix Figure . PrelimiRing-finger And WD40 associated Ubiquitin-Like) domain.In summary, considering the profile-profile comparisons E-values Figure , secondaThe ubiquitin related proteins fall into two separate classes . Ubq-lik1aar) as K29, K48, and K63). Hence, it appears unlikely that this domain functions as a traditional UBL modifier. PRC1 Ring finger and WDR48-p80 proteins therefore could be included in the group of UDPs. There are two classes of UDPs as well [RAWUL domain lacks the characteristic C-terminal diglycine motif required for enzymatic conjugation of the Ubq domain to an acceptor lysine. Neither are conserved the three most common acceptor lysine residues involved in polyubiquitination  proteins have the Ubq-like domain located at or close to the N-terminus of the protein and defined by a stretch of 45–80 residues with significant sequence homology to ubiquitin, while the Ubiquitin regulatory X (UBX) domain proteins have the Ubq-like domain at the C-terminus consisting of ~80 amino acids that shares common secondary structure organization with ubiquitin despite the lack of significant sequence homology ,65. The Conversely, only very few UBX domain proteins have been studied in detail and no general function for the UBX domain has yet emerged. Most of the UBX domain proteins identified so far can be grouped into four evolutionary conserved protein families represented by the human Fas-associated factor-1 (FAF1), p47, Y33K, and Rep8 proteins . Recent In the case of the poorly characterized WDR48-p80 family -52, the On the other side, our analysis revealed that the combination of a Ring finger domain at the N-terminus and a RAWUL domain at the C-terminus is a key feature to define the PRC1 Ring finger protein family, since no other protein presents this domain architecture. The Ring finger domains of the Ring (Ring1A and 1B) and Bmi1 proteins are responsible for the E3 ubiquitin ligase activity ,34, wherOryza sativa, Vitis vinifera and worms that share the PRC1 ring finger domain architecture, an N-terminal Ring finger domain and a RAWUL domain at their C-terminal region  shows that Arabidopsis Like Heterochromatin Protein1  is functionally similar to Pc, a subunit of PRC1 that recognizes and binds the H3K27 marks created by PRC2. Similarly, PRC1 Ring finger proteins were thought to be missing in plants; however, our analysis revealed the presence of putative orthologs to these proteins. All together these data suggest that PRC1 is also conserved in plants, in agreement with our analysis.Based on sequence similarity and biochemical data, two groups of PRC1 Ring finger proteins have been defined ,36, the C. elegans, PcG-mediated gene repression has been proposed to require a PRC2-like complex [C. briggsae [C. elegans [C. elegans and in C. briggsae (Figure Similarly, lower metazoans, such as nematodes, are also thought to lack PRC1 homologs. In  complex  and two  complex ,28. Desp complex ,28. Howe elegans , indicate Figure . The funWe detected statistically significant sequence similarity between the ubiquitin superfamily and the conserved C-terminal regions of PRC1 Ring and WDR48-p80 families, defining a new Ubq-like domain, the RAWUL domain. The identification of an Ubq-like domain in the PRC1 Ring family offers new experimental approaches aimed at elucidating their roles in important cellular processes, such us, stem cell self-renewal and cancer. In addition, characterizing the RAWUL domain allowed us to identify putative PRC1 Ring finger proteins that were thought to be missing in the plants and worms. The possibility that a PRC1-like complex is also involved in PcG-mediated gene silencing mechanism in these organisms is intriguing and opens new avenues in PcG investigation.We first performed BLAST sequence similarity searches  against 1gnu[Secondary-structure predictions were performed with Predict Protein server . Fold-re1gnu. Models 1gnu. The fig1gnu.LSP carried out the database search, sequence analysis and drafted the manuscript. DD carried out the protein structure prediction and modelling, and helped to draft the manuscript. ZRS participated in the coordination and helped to draft the manuscript. MC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Homology model of the RAWUL domain from human Bmi1 protein.Click here for fileWeb page with additional information at: Click here for file


Ribonuclease P is an ancient endonuclease that cleaves precursor tRNA and generally consists of a catalytic RNA subunit (RPR) and one or more proteins (RPPs). It represents an important macromolecular complex and model system that is universally distributed in life. Its putative origins have inspired fundamental hypotheses, including the proposal of an ancient RNA world.To study the evolution of this complex, we constructed rooted phylogenetic trees of RPR molecules and substructures and estimated RPP age using a cladistic method that embeds structure directly into phylogenetic analysis. The general approach was used previously to study the evolution of tRNA, SINE RNA and 5S rRNA, the origins of metabolism, and the evolution and complexity of the protein world, and revealed here remarkable evolutionary patterns. Trees of molecules uncovered the tripartite nature of life and the early origin of archaeal RPRs. Trees of substructures showed molecules originated in stem P12 and were accessorized with a catalytic P1-P4 core structure before the first substructure was lost in Archaea. This core currently interacts with RPPs and ancient segments of the tRNA molecule. Finally, a census of protein domain structure in hundreds of genomes established RPPs appeared after the rise of metabolic enzymes at the onset of the protein world.The study provides a detailed account of the history and early diversification of a fundamental ribonucleoprotein and offers further evidence in support of the existence of a tripartite organismal world that originated by the segregation of archaeal lineages from an ancient community of primordial organisms. With few exceptions , ribonucThe ubiquitous distribution of RPR molecules in life suggests that a primordial RPR form was already present before the diversification of the three superkingdoms of life, Archaea, Bacteria, and Eukarya . FurtherBacillus subtilis indicates substrate specificity can be altered without changing the basic cleavage reaction [Altman and Kirsebom  proposedreaction . In factreaction . This mireaction -22. Howereaction -25. Furtreaction . All theHere we study the evolution of the RNase P complex with a well-established phylogenetic method that reconstructs evolutionary history directly from structure . This clWe illustrate our cladistic approach with a flow diagram that describes how we study RNA and proteins Figure . When anNEXUS format (as is or transposed), which are then used as input text file for equally weighted unconstrained maximum parsimony (MP) analysis. In the file, the first column represents a phylogenetic character and individual numerical values character states. Rows are phylogenetic taxa. The input files generate rooted trees of molecules or trees of substructures depending on what is considered taxa  and this determines the preferred direction (polarity) of character state change. This hypothetical ancestor in our example has the maximal character state for each character and is included in the search for optimal rooted trees with the sole purpose of polarizing character state transformation. No external hypotheses in the form of outgroups  are needed to root the trees. The external hypotheses are replaced by the more axiomatic polarization assumption of our evolutionary model. For more detailed descriptions of the method and model, see Materials and Methods and for example [We illustrate the analysis in more detail with a concrete example of how we build trees of molecules and trees of substructures Figure . We firs example -33,41.With proteins, we use hidden Markov models (HMMs) of structural recognition to survey protein sequences in genomes, decomposing proteins into protein domains at FSF level Figure . The surRecent studies have indicated that RPR molecules are suitable for phylogenetic analysis of closely related bacterial taxa and have potential as a tool for species discrimination . One disDetailed phylogenetic patterns were maintained when data were partitioned according to superkingdoms or according to sequence, structure, or structural domain Table . As prevIn Archaea, the monophyly of Crenarchaeota was recovered by the combined analysis of structure and sequence data Figure  but not Escherichia coli, is found in most bacterial and archaeal organisms. The Bacillus-type or type B, represented by Bacillus subtilis, is found only in low-GC Gram-positive bacteria [Thermomicrobium, a member of the green non-sulfur bacteria, to form type C RPRs  in comparison to the ancestral type A structure [An accurate model for RPR structure is a prerequisite for understanding the mechanism of substrate recognition (S domain), catalytic activity (C domain), and evolution of the ribozyme, and is supported by a significant body of evidence from biochemical, photochemical, molecular, crystallographic, and phylogenetic comparative studies. For example, refined secondary structure models have been inferred by identifying concerted changes (covariation) in the nucleotide sequence of RPRs that share common ancestry and function and have been generally confirmed by biochemical and crystallographic evidence -59. In tbacteria . Throughtructure ,64. FinaTrees of molecules dissected the evolutionary history of the different types of RPR structures and their evolutionary origin Figure . MappingThermomicrobium roseum was placed at the base of trees of molecules  provide a chronology that establishes which parts of the molecule are ancestral and which parts are derived. We generated trees describing the evolution of helical stems, hairpins, bulges, and unpaired regions Figure  constituThe most ancient substructure in the tree was P12, a terminal stem of the S domain Figure . This suMycoplasma fermentans and Thermoproteaceae, species with highly reduced genomes [The taxonomic distribution of evolving stem substructures revealed that, with an exception in the P8 stem that was lost in type M archaeal molecules, the first 10 most ancient structures were universally present in all RPR molecules Table . Note ho genomes ,72. The With the exception of stem P12, the C domain was in general more ancient than the S domain on all of the trees of stem substructures analyzed. The ancestral nature of the C domain was also revealed in trees of other substructures  in the RPR molecule Figure  and the in vivo. In addition to pre-tRNAs, there are a few other substrates for RNase P in vivo, such as pre-4.5S RNA, pre-tmRNA, a few mRNAs, and riboswitches [It is critical that we examine RNA-protein interactions in the RNase P complex, given that proteins and catalytic RNA are both required for enzyme activity switches -78. To dswitches ,79-82.Thermotoga maritima) and type B (Bacullus stearothermophilus) RPRs [Bacterial RNase P studies show that type B and ancestral type A molecules can interchangeably activate RNA catalytic functions ,71,82,83us) RPRs ,58,59 reus) RPRs ,84,85. Tus) RPRs ,82,86.As discussed previously, our results support the hypothesis that the C domain is the ancestral structural and functional domain . HoweverPyrococcus furiosus interact with the S and C domains, respectively, enabling the inter-domain cooperation required for optimal pre-tRNA recognition and catalysis. Enzymatic footprinting also demonstrated that the RPP21/RPP29 protein complex in Pyrococcus furiosus interacts only with the S domain of the RPR [Saccharomyces cerevisiae also suggests some likely functions of P3 in stabilizing the enzyme's structure and in interactions with pre-tRNA [S. cerevisiae RNase MRP and RNase P, Pop6 and Pop7 form a heterodimer that binds directly to P3, protecting a segment of the lower strand of the internal loop of P3 and part of the adjacent helical stem [S. cerevisiae RNase P and RNase MRP [It is noteworthy that both archaeal and eukaryal RPPs show extensive protein-protein and protein-RNA interactions, and that some of these may also involve the S domain. Furthermore, only a subset of the protein subunits may be necessary for catalytic activity while other proteins function in assembly and/or localization. In fungi, only two (Pop1p and Pop4p) out of nine nuclear RPPs interact with RPR . Tsai et the RPR . Moreovepre-tRNA . In humapre-tRNA . In S. ccal stem . Similarcal stem . Furthercal stem . For exaNase MRP .The number of proteins associated with catalytic RNA varies greatly between Archaea, Eukarya, and Bacteria. In general, there are 4-5 and 9-10 protein units in archaeal  and eukand = 0.005), and corresponded to the recently identified RPPs that do not associate with RNA cofactors [nd = 0.06 in the ribosomal protein S5 domain-like domain (d.14.1) typically found in bacterial complexes to nd = 0.803 in the AlbA-like domain (d.68.6).In order to study the evolution of RPPs and determine the putative age of protein-RNA contacts, we timed the appearance of the 3D structure of RPP-associated domains in a tree of architectures derived from phylogenomic analysis of domain structure at FSF level of structural classification Figure . The gloofactors . The agend = 0.06), the ribosomal protein S5 domain-like FSF (d.14.1) depicted in the complex of Figure We previously identified three epochs in the evolution of proteins  and RNA nd = 0.005). This suggests that the addition of the RPR moiety to the catalytic protein-based RNase P enzymes is either a derived feature or alternatively that the domain was co-opted late in evolution in eukaryotic organelles to perform the ribonucleoprotein task. However, the fact that the make-up of RPP domains is varied and evolutionarily diverse in RNase P suggests recruitment plays an important role in evolution of catalysis in this complex and the argument can be used to disfavor the idea that organellar protein enzymes are ancient fossils. Without additional evidence capable of dissecting recruitment, however, the age of the c.2.1 domain cannot be used to support or refute the ancestrality of RPPs relative to RPRs.One interesting observation is the age of the domain linked to RNase P enzymes in organelles of Eukarya that do not require RPR cofactors  . The domStudies have shown that the S domain contributes to pre-tRNA recognition and helps position the substrate for optimal cleavage ,102,103.Results also underscore the significance of a primordial stem-loop that originally harbored a multitude of primordial functions, most of which were lost or displaced as molecules evolved and gained specific roles. The concept of a hairpin being the starting component of tRNA  has beenOur study reveals several important evolutionary patterns linked to the structure and function of RNA and protein components of RNase P: (i) the early origin of archaeal RPR molecules, which suggests the lineage leading to superkingdom Archaea is ancestral; (ii) the origin of the RPR molecule in the P12 substructure, closely followed by the catalytic P1-P4 core structure; (iii) the ancient origin of the C domain; (iv) the early appearance of RPP substructures that interact with proteins in this primordial RNase P complex; (v) and the ancestral nature of RPR-associated proteins, which originated at the onset of the protein world. Based on these observations we propose a model for the early evolution of the ribonuclease catalytic complex in a lineage leading to the last universal common ancestor of life Figure . In thishttp://jwbrown.mbio.ncsu.edu/RNaseP/home.html; Release No. 12, 2005 edition, [Entamoeba histolytica [Zea mays [Candidatus Kuenenia stuttgartiensis [Reclinomonas americana), two chloroplast (Porphyra purpurea and Zea mays), and one cyanelle (Cyanophora paradoxa) sequences were closely related to bacterial sequences, a result that is consistent with the evolutionary origins of these organelles. These four taxa were subsequently included in Bacteria. Table The secondary structures of 130 RPR sequences were retrieved from the RNase P Database . Partiaedition, . Anothertolytica , Zea mayZea mays , and Canrtiensis  were alsOverall, we scored a total of 129 structural characters in the 133 RPR molecules analyzed by comparative sequence analysis, comparison with crystallographic models, and other criteria  or the branching, stability, and plasticity  of homologous substructural components. In this study we focus on the former. Homologous substructures represent those that are of the same kind  and respond to the same evolutionary model defining the character transformation sequences. For example, we reconstruct trees of coaxial stems corresponding to the helical regions in RPR, separate from trees of hairpin loops. This is because character change leading to coaxial stem taxa depends on models of character state that are quite different from those governing unpaired segments.Structural features were treated as linearly ordered multistate characters that were polarized by invoking an evolutionary tendency towards molecular order. Establishing a preferred directionality of character state change resulted in intrinsically rooted trees, which were then used to define lineages that are either ancient or derived. Operationally, polarization was determined by fixing the direction of character state change using a transformation sequence that distinguishes ancestral states as those thermodynamically more stable. Maximum character states were defined as the ancestral states for stems and G:U base pairs (i.e. structures stabilizing the RPRs). Minimum states (0) were treated as the ancestral states for bulges, hairpin loops, and other unpaired regions (i.e. structures de-stabilizing the RPRs). The validity of character argumentation has been discussed in detail elsewhere ,41,115, Many studies that focus on molecular mechanics strongly support a tendency towards molecular order. Comparative studies of extant and randomized sequences show that evolution enhances conformational order and diminishes conflicting molecular interactions ,116-122.in vitro evolution of ribozymes [A molecular tendency towards order can be linked to fundamental concepts in thermodynamics . The "buibozymes .A tendency towards order is also supported by dissipation tendencies in energy and matter that exist in an open cosmological model of the Friedmann type . This moFinally and more importantly, tendencies towards structural order are experimentally supported by phylogenetic congruence of phylogenies reconstructed using geometrical and statistical structural characters ,33,34 anMULTREES option selected. One shortest tree was saved from each search. Hypothetical ancestors were included in the searches for the MP trees using the ANCSTATES command. A "total evidence" approach [5 replicate analyses using "fast" stepwise addition of taxa in PAUP*. The g1 statistic of skewed tree length distribution calculated from 104 random parsimony trees was used to assess the amount of nonrandom structure in the data [Data matrices were analyzed using equally weighted MP as the optimality criterion in PAUP* . Note thapproach ,141, alsapproach , was appapproach  were calthe data .evolutionary heat maps of ancestry. Because reconstructed trees were intrinsically rooted, we established the relative age (ancestry) of each substructure by measuring a distance in nodes from the hypothetical ancestor on a relative 0-1 scale. To do this, we counted the number of nodes in every lineage from the root to the terminals of the tree and divided this number by the maximum number of nodes in a lineage [Evolutionary relationships derived from trees of substructures were traced in generic 2-dimensional models of RPR secondary structure that we here call  lineage . AncestrSUPERFAMILY and a probability cutoff E of 10-4. FSFs were defined by the SCOPhttp://scop.mrc-lmb.cam.ac.uk/scop/ version 1.69 [http://www.uniprot.org/ and downloaded 1,136 protein sequences in Fasta format. HMMs were then used to predict the SCOP identifiers (IDs) describing individual FSFs linked to the sequences. We finally used the "SCOP parseable files" link in SCOP to identify the corresponding IDs in the "dir.des.scop.txt 1.69" file. PROTEIN DATA BANK (PDB) files associated with RPPs were queried and downloaded from the PDB database http://www.rcsb.org/pdb/home/.A census of the genomic sequence of 584 organisms, including 46 Archaea, 397 Bacteria and 141 Eukarya, assigned protein structural domains corresponding to 1,453 fold superfamilies (FSFs) to protein sequences using advanced linear hidden Markov models (HMMs) of structural recognition in ion 1.69 . The cenion 1.69 . The recion 1.69 . A recenion 1.69 . In ordeBoth authors conceived, designed, and performed experiments, analyzed the data, and wrote the article.Figure S1 Phylogenetic trees of RPR molecules derived from the C domain. Figure S2 Phylogenetic trees of RPR molecules derived from the S domain. Figure S3 Phylogenetic trees of RPR molecules derived from a conserved substructural core. Figure S4 Phylogenetic trees of RPR molecules derived from a conserved substructural core. Figure S5 Phylogenetic trees of stem substructures derived from the C and S domains of the RPR molecule. Figure S6 Cumulative frequency distribution plot of molecular substructures. Table S1 Structural characters and their statistics (range and mean ± standard deviation) used in phylogenetic analyses. Table S2 Data matrix of structural characters used in the cladistic analyses for RPR molecules.Click here for file