LCL domain catalyzes a peptide bond between two L-amino acids, a DCL domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β-hydroxy-carboxylic acid , and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An DCL and Starter domains) and their characteristic sequence differences, mutually and in comparison with LCL domains. Based on their phylogeny and the comparison of their sequence motifs, LCL and Starter domains appear to be more closely related to each other than to other subtypes, though pronounced differences in some segments of the protein account for the unequal donor substrates (amino vs. β-hydroxy-carboxylic acid). Furthermore, on the basis of phylogeny and the comparison of sequence motifs, we conclude that Dual E/C and DCL domains share a common ancestor. In the same way, the evolutionary origin of a C domain of unknown function in glycopeptide (GP) NRPSs can be determined to be an LCL domain. In the case of two GP C domains which are most similar to DCL but which have LCL activity, we postulate convergent evolution.In this paper, we report on the reconstruction of the phylogenetic relationship of NRPS C domain subtypes and analyze in detail the sequence motifs of recently discovered subtypes  for the sequence motifs as well as for the entire sequences. The determined specificity conferring positions will be helpful for the mutation of one subtype into another, e.g. turning  Scientifically, it is a challenge to discover how these structurally complex macromolecules are synthesized by the concerted interworking of the multi-domain proteins NRPS and polyketide synthases (PKS) that synthesize a peptide or ketide backbone with several other modifying and "decorating" enzymes . NRPS belong to the family of megasynthetases, which are among the largest known enzymes with molecular weights of up to ~2.3 MDa  [The biologically active products synthesized by non-ribosomal peptide synthetases (NRPSs) are of interest for a variety of reasons: Pharmaceutically, a rich collection of them are used as drugs like antibiotics (e.g. penicillin and vancomycin), anti-tumorals and cytostatics (e.g. bleomycin), anti-inflamatorials and immunosuppressants (e.g. cyclosporin A), toxins . Chain elongation is terminated by the action of a thioesterase (TE) domain. It is usually the final domain of the last module in the assembly line and catalyzes either the hydrolysis or the intramolecular cyclization of the peptide chain, yielding a linear or macrocyclic product [Actinomycetales is known for the wealth of secondary metabolites produced by its members and comprises, among others, Streptomyces species, Corynebacteria and Mycobacteria. The majority of all currently known antibiotics and other therapeutic compounds are derived from Streptomycetes [Corynebacteria and Mycobacteria are human pathogens which produce toxins as secondary metabolites. The structural and functional diversity of non-ribosomal peptides, unlike ribosomally synthesized peptides, arises from the incorporation of unusual amino acids: During the assembly of the peptide backbone by the NRPS, both proteinogenic and non-proteinogenic amino acids (e.g. ornithine), including D-amino acids, may be integrated and modified "on-the-fly" by enzymatic domains within the NRPS protein. Possible  modifications of the building blocks (= amino acids) are N-acylation of the first amino acid, epimerization (into D-amino acids), N-methylation, or cyclization of amino acids  with an amide-nitrogen of the peptide "backbone", resulting in oxazolines (e.g. in vibriobactin) and thiazolines (e.g. in bacitracin); these can be further oxidized or reduced by special domains [The adenylation (A) domain specifically recognizes one amino acid (or hydroxy acid) and activates it first through the formation of an aminoacyl adenylate and then via covalent bonding of the activated amino acid as a thioester to the 4'-phosphopantetheinyl (4'PPant) cofactor of the peptidyl carrier protein  domain). The third compulsory domain is the Condensation (C) domain, which catalyzes the elongation reaction of the peptidyl chain tethered to the phosphopantetheinyl arm of the upstream T domain to the amino acid bound to the downstream T domain . This iomycetes . Many me domains , and fur domains . Further domains .et al. [In this paper, we report on the functional variants (subtypes) and homologues of the Condensation (C) domain of NRPS. All C domain sequences of this study were extracted from NRPS that were detected in all available completely sequenced bacterial genomes and a comprehensive collection of annotated biosynthesis clusters. Besides A domains  C domaiet al.  demonstret al. [Because C domains have been shown to have non-negligible specificity for the amino acid that is activated by the downstream A domain, swapping whole modules or insertion/deletion seems to be more promising, provided that the integrity of the functional domains is carefully maintained and the modules are dissected in their linker regions ,9. Neveret al.  for the Furthermore, we have analyzed C domains and Epimerization (E) domains of glycopeptide NRPS. In these proteins, two Condensation domains preceded by former (now inactive) Epimerization domains have gained opposite stereoselectivity, probably due to convergent evolution, for which we accumulate evidence. Additionally, we discuss the origin of a C domain (often referred to as X* domain) at the C-terminus of glycopeptide NRPS, which is thought to be inactive.The C domain has two binding sites: one for the electrophilic donor substrate (the acyl group of the growing chain) and one for the nucleophilic acceptor substrate (the activated amino acid). The condensation reaction involves catalysis of a nucleophilic attack by the amino group of the aminoacyl adenylate bound to the downstream PCP on the acyl group of the growing peptide chain which is bound to the upstream PCP ,11. The In particular, C domains succeeding an E domain are expected to show specificity towards the configuration (L or D) of the C-terminal residue that is bound at the donor site because the preceding E domain does not specifically catalyze the epimerization from L to D but provides a mixture of configurations. It is the role of the C domain to select the correct enantiomer . MoreoveDCL-catalysts because of this behavior [C domains immediately downstream of E domains were shown to be D-specific for the upstream donor and L-specific for the downstream acceptor, thus catalyzing the condensation reaction between a D- and an L-residue. These C domains were termed behavior .LCL-catalysts promote the condensation of two L-amino acids. Both LCL- and DCL-catalysts possess a conserved His-motif in their active site. The consensus sequence of this motif is HHxxxDG where x denotes any residue  domains have recently been identified. This finding was based on the observation of NRPS which had products that contained D-residues although the NRPS itself did not show an E domain in the corresponding module. Biochemical experiments supported the hypothesis that Dual E/C domains exist which are activity . In the activity   domains which catalyze both peptide bond formation and subsequent cyclization of cysteine (Cys), serine (Ser), and threonine (Thr) residues. The five-membered heterocyclic rings which result from this reaction are important for chelating metals or interaction with proteins, DNA or RNA. Cyc domains are structurally related to C domains and are supposed to be evolutionary specialized C domains . In Cyc et al.  found th1. condensation performed by ordinary C domains; 2. condensation and subsequent heterocyclization catalyzed by Heterocyclization (Cyc) domains; 3. epimerization followed by condensation which are both catalyzed by a Dual E/C domain; 4. Starter domains (see below) which are found on initiation (= first) modules and acylate the subsequent amino acid.A total of 481 Condensation domains  were extracted from 182  NRPS and 31 NRPS/PKS hybrid sequences found in 62 bacterial genomes out of the 256 bacterial genomes screened, employing pHMMs as described in Section Methods . Altogether 108 C domains were obtained from 42 NRPS sequences from gene clusters downloaded from the UniProt database. After removing doublets, all 525 non-identical C domains and homologues obtained were multiply aligned and phylogenetic trees were built. The resulting tree topology was clearly dominated by the functional categories that are known for C domains (as described in the previous section), rather than species phylogeny or substrate specificity alone. The four main functions are: LCL-catalysts and DCL-catalysts according to the stereochemistry of their substrates. The existence of all these functional subtypes is reflected by the phylogeny. Fig. Ordinary C domains may further be classified into LCL- and DCL-catalysts were either distinguished according to their domain structure or by their position in the phylogenetic tree. By this, 275 domains of all 525 C domains were classified as being LCL-catalysts, 69 were DCL-catalysts and 42 were Starter C domains (see next section).For further analysis, the different subtypes were examined separately. While Cyc and Dual E/C domains could be identified by means of their characteristic sequence motifs , When analyzing the Condensation (C) domain phylogeny, it became apparent that some domains did not cluster with the known C domain subtypes. A closer look at the location of these deviating C domains revealed that all of them were the very first C domain of the corresponding NRPS assembly line. The remaining C domains of these assembly lines appeared in other subtrees in the phylogeny.β-hydroxyl fatty acid which is connected to the first amino acid of the peptide chain [Included in this set of starter C domains are those stemming from the biosynthesis clusters for the lipopeptides surfactin , lichenyde chain . The pepde chain . This acde chain  and fengde chain . Moreovede chain .Streptomyces coelicolor [Mycobacterium avium [α-amino acid. Assured by the fact that these Starter C domains match significantly well to the profile HMM built from the Starter C domain sequences that process β-hydroxy fatty acids, we compared salicylic acid with β-hydroxy fatty acids. Because both are β-hydroxy-carboxylic acids with no amino-substituent at the α position, as a-amino acids would have, we assume that this is the structural characteristic recognized by the prototype of Starter C domains. The profile HMM built from all Starter C domains in our data set (together with the pHMMs of the other domains) presents a powerful instrument for exploring and understanding tricky NRPS domain-product relations.The same has been observed for the synthesis of the acidic lipopeptide CDA in elicolor  and the um avium . The Staum avium . SnbA isum avium . A similum avium . In concNote that Formylation domains as found, for example at the N-terminus of linear gramicidin synthetase subunit A  are not et al. [et al. [et al. [DCL domains [The different core motifs in Condensation domains have first been described by de Crécy-Lagard et al.  and reco [et al.  but have [et al.  but the  domains ,30 as we domains  domains et al. [et al. [LCL sequences, in which "predicted" means that they were classified based purely on their position in the phylogenetic tree while "verified" sequences were checked individually taking into account their position in the succession of neighboring NRPS domains, the presence of discriminative unique motifs (see Methods Section) and/or literature information. For the DCL motifs, 23 verified and 46 predicted sequences were used, 7 verified and 35 predicted for the Starter domains, and domains 9 verified and 47 predicted for the Dual E/C domains.The sequence motifs represented in Fig. et al.  which, aet al.  which ma [et al. , and alsBased on three publications, four residues are likely to be essential for the catalytic activity of the C domain. The most important residue is the 2nd His of the active site His-motif .Furthermore, six residues have been identified as being structurally important or as playing a role in correct folding of the domain. In the following, these residues are presented, grouped by their role  with respect to (w.r.t.) VibH ,33,34et al. [LCL, DCL or Starter.#9 Trp264 (W) is catalytically important in VibH according to Keating et al. , but the#10 Asn335 (N) w.r.t. VibH [#6 Gly131 (G of the active site His-motif) w.r.t. VibH #1 Arg62 (R) w.r.t. TycB1 #5 Asp130 (D) w.r.t. VibH ,33,34#8 Arg263 (R) w.r.t. VibH  = Arg278#2 Arg67 (R) w.r.t. TycB1 #3 His146 w.r.t. TycB1 (1st His of active site His-motif) #7 Trp202 (W) w.r.t. TycB1 LCL and DCL domains do not differ significantly in any of the residues identified as being of catalytic or structural importance (except residues Nb. 9 and Nb. 10). However, using methods described in Section Methods, 20 positions in which LCL and DCL have significant differences according to SDPpred [LCL and DCL subtypes. The same is true for the region downstream of C4  where a moderately conserved motif LPxDxxRP is seen in LCL which is completely absent in DCL  domains but show no conservation among Starter C domains. When we compare C domain sequence motifs, it is apparent that motifs C2 and C4, despite being well conserved in LCL, are unconserved in Starter domains, which presumably can be explained by the much broader structural range of substrates processed by Starter domains.While not being conserved at residues Nb. 5, Nb. 7, Nb. 9, and Nb. 10, all remaining 6 functionally important residues are highly conserved throughout the putative Starter domains. When comparing LCL and Starter C domains are more closely related to each other than to other subtypes . Furthermore the phylogenetic tree shows that Dual E/C and DCL domains share a common ancestor. We tested the reliability of the phylogenies depicted in Fig. LCL and Starter C domains as well as of Dual E/C and DCL domains. In addition, we generated an alignment using DIALIGN [DCL domains are grouped together as are LCL and Starter C domains.The reconstructed phylogeny of C domain subtypes reveals that see Fig. . Comparisee Fig.  account  DIALIGN , which iDCL domains are very similar to each other and dissimilar to LCL and Starter domains. This observation of the relationship between the four subtypes is consistent with the stereochemistry of the substrates, bearing in mind that Dual E/C domains function as DCL because the substrate L-amino acid is first epimerized by the intrinsic epimerization activity of the domain [Especially in motif C5, Dual E/C and e domain .DCL and LCL domains, the tree topology reflects the species phylogeny of the bacteria rather than substrate specificity of any kind. We analyzed this by reconstructing phylogenies for DCL domains and LCL domains separately to be able to see the topology within these subtypes in more detail (data not shown). The reconstructed phylogenies did not give any evidence that would support the hypothesis that C domains cluster according to their specificity towards the condensated amino acids. This analysis, however, is based on the complete C domain sequence. A strategy to investigate whether C domains exhibit substrate specificity would involve predicting putative specificity determining positions using entropy and/or conservation based approaches , or inferring of putative active site residues by homology with the VibH structure  and M3 and M6 in complestatin (com) clusters which contain an E domain not present in the other clusters. The M3-E domain, however, is assumed to be inactive [com M6 has not been reported elsewhere so far. We were able to detect it with an hmmpfam scan using the specific E domain pHMM. All six NRPSs contain a domain X* of unknown function. Until now, it has been characterized as an atypical C or E domain but its role in glycopeptide synthesis remains to be clarified. In general, it is assumed that the stereochemistry of a NRPS product can be predicted from its domain structure. In the case of the known glycopeptides, the domain organization implies the stereochemistry NH2-L-D-L-D-D-L-L-COOH, provided that the E in module M3 is inactive and that the X* domain does not function as an E domain. This stereochemistry is inconsistent with the chemically determined structure of the products: NH2-D-D-L-D-D-L-L-COOH [cep cluster, however, Trauger and Walsh [All these NRPSs comprise seven modules. They show an identical domain composition, with the exceptions of module M3 in the A47934  it is followed by remnants of an adenylation domain , the loss of the functional E domains in M3 and M6 would have been a selective gain. Comparing all M4 and/or all M7 C domains with all DCL domains using SDPpred did not reveal any significant positions; comparing them against the other glyco-C domains gave thirty positions. As all glyco-C domains are very closely related and differences between them might also reflect substrate selectivity (not only stereo-selectivity) or different inter-domain interacting residues, we cannot decide which of them confer the altered stereo-selectivity. One point to notice however, is a (positively charged) His in all M4 glyco-C domains at position 6 in the extended motif C2 where an (uncharged polar) Gln is highly conserved in other DCL domains. This position has also been selected by FRpred as a significant (= subtyping) position. The other positions do not represent mutations in highly conserved residues (data not shown). It would be necessary to check their significance experimentally with mutation studies. It would also be helpful to compare the peculiar sequences with more glyco-C domains, but others are -unfortunately – not publicly available.The assumption that the diverged C domains of modules M4 and M7 would have adopted mutations at positions that we have previously determined as "specificity determining positions" was disproved. Probably, a few spontaneous mutations in the DCL to LCL in glyco-C domains, interesting experimental questions can be formulated based on our findings. For example, one could think of mutational studies with the goal of altering the stereo-selectivity of a DCL domain and to determine the relevant residues experimentally. A starting point could be, for example the M6 C domain of any glyco-NRPS.However, although we could not discover which altered positions are responsible for the functional shift from The second His of the His-motif in motif C3 which is important for catalysis is replaced by Arg (R). Also, the Gly of the His-motif is not present but replaced by Arg in all but one X* domain. Note, however, that while the second active site His is invariant in C domains, Gly138 is not.LCL-domains of Streptomyces species. Only three of these coincide with residues of functional importance: His126, Arg278 and Asn335. Furthermore, a C terminal region could be detected in which M7-X* and LCL differ strikingly. The concordance of M7-X* with the most highly conserved residues of Streptomycete LCL domains supports the phylogenetically based suggestion that M7-X* is an inactive LCL domain.SDPpred predicted 13 specificity determining residues when comparing M7-X* to LCL domains, which condense two L-aminoacids, DCL domains, which condense a D-amino acid  with an L-amino acid, and Starter C domains (an expression that we coin here) which connect a β-hydroxy-carboxylic acid (e.g. β-hydroxyl fatty acid) with an L-amino acid. The phylogeny of C domain homologues is reconstructed using NRPS sequences (including hybrid NRPS) from completely sequenced genomes (43 genomes contained NRPS) and selected biosynthesis clusters, involving 525 non-identical C domain sequences. The sequence motifs of LCL, DCL and Starter domains have been extracted and are presented as sequence logos: for LCL domains, this represents an update of consensus sequences published by Marahiel et al. [DCL and Starter domain motifs are analyzed and mutually compared for the first time. For comparison, the homologous motifs are also presented for Dual E/C domains, which were first described by Balibar et al. [In this study, we present the evolutionary relationship of homologues of the NRPS Condensation domain which include enzymatic domains catalyzing Epimerization, Heterocyclization, Condensation and Epimerization with subsequent Condensation in one domain . The Condensation domain itself appears in three subtypes according to the stereo-chemistry of the substrates catalyzed: l et al. ; DCL andr et al. .DCL subtree of the phylogenetic tree and show all DCL sequence motifs, although they clearly have LCL activity. This suggests that they might be an example of convergent evolution. Even though this is probably a rare event, its possibility has to be kept in mind when uncharacterized C domains are to be classified, e.g. using profile HMMs provided as Additional files LCL domains and is followed by remnants of an A domain, implying an additional complete module in the ancestor of glyco-NRPS.We have investigated the "mysterious" evolutionary origin of C domains in glycopeptide antibiotic synthesis clusters and have discovered that two of the six C domains present in these glyco-NRPSs appear in the et al. [Roongsawang et al.  have alr. In total, the genomes of 256 bacterial species were downloaded and screened for NRPS protein sequences (including NRPS/PKS hybrids). Additional protein sequences of PKS and NRPS which are part of known secondary metabolite biosynthesis clusters were obtained from the UniProt database [Actinomycetes and one from Pseudomonas , which are statistical models extracted from multiple sequence alignments. In contrast to simple sequence motifs of fixed length, i.e. position specific scoring matrices, pHMMs are suited for identifying motifs that are interrupted by segments of variable length, and are used to characterize position-specific sequence similarities within a family of proteins. A collection of pHMMs for a wide array of domains and domain families is availabe from the database Pfam  and TIGRet al. [et al. [The Pfam pHMM Condensation (PF00668) recognizes both the Condensation (C) and Epimerization (E) domain of NRPS. The intention, however, is to be able to distinguish between these two domain types. Therefore C domain and E domain specific pHMMs were generated from a multiple sequence alignment (MSA) of Epimerization domains and non-Epimerization domains, both of which were recognized by the Pfam C pHMM. To obtain a set of Epimerization domains, all NRPS sequences with complete modules were extracted from all bacterial protein sequences in the Uniprot database  as descret al.  and revi [et al. ). Phylog [et al.  and chec [et al. ,55.The quality of a reconstructed phylogenetic tree crucially depends on the underlying multiple sequence alignment. All sequence alignments in our study were generated using MUSCLE ,53. The C domains catalyze the condensation of two amino acids, thus, they have two binding sites: the acceptor and the donor site. To be able to investigate whether the substrate specificity of one of these sites influences the phylogeny of the domain, the specificity of the preceding and succeeding A domain in the assembly line was predicted with the NRPSpredictor  and storLCL- or a DCL-catalyst was established according to the observed domain organization of the modules in an NRPS sequence  domain in the protein. In this case, the type of the condensation reaction can only be assigned if the order in which the proteins act in the assembly line is known. To overcome this problem, we checked all assignments with the classification suggested by the phylogeny.Functional subtypes may be distinguished on the basis of sequence features, domain architecture or clustering behavior during tree reconstruction. Condensation and Heterocyclization domains may be discriminated by the sequence motif they exhibit at their active site. The occurrence of a sequence motif within a longer sequence can be detected with the help of a position specific score matrix (PSSM) . PSSMs wo et al. ). It is If the order of the subunits is unknown, temporarily incorrect assignments can only be revised later in the analysis.et al. [et al. [et al. [et al. [In a set of homologous enzymes, we may find subsets that each contain sequences with one distinct substrate specificity. These subsets of common function are called subtypes and often vary at certain positions, whereas the same positions may be conserved within a given subtype. Li et al.  call the [et al.  refer to [et al.  and Kali [et al. . For thi [et al. . Here, t [et al. ,57 whichSeveral methods were applied for reconstructing phylogenetic trees from the multiple sequence alignments that were generated for each domain type. Trees presented in this article were reconstructed using protein sequences, as amino acid sequences are preferred to nucleotide sequences because they are more conserved and are not influenced by compositional bias like G+C content and codon usage. In addition, the mathematical model for the evolutionary change of amino acid sequences is much simpler than that of nucleotide sequences, which reduces the risk that the phylogeny is based on wrong evolutionary assumptions, since just a suitable substitution matrix has to be selected . The amiIn some cases, the rate of amino acid substitution may be assumed to be the same for all positions in the alignment. In general, however, this does not reflect reality since the substitution rate is usually higher at positions of lower functional importance. A more realistic model is achieved if the substitution rate is taken to vary among sites according to the gamma distribution .α which determines the shape of the Γ distribution as an option. Whenever a gamma distributed rate variation was assumed, four gamma-rate categories were used to approximate the distribution. Several tree reconstruction methods were applied to each dataset to determine whether different methods yield different topologies, which in turn would indicate that the proposed topologies are unreliable. As a distance-based method, the Neighbor-Joining (NJ) method [Apart from PHYLIP , all met) method  was appl) method  and PHYM) method  were appBootstrapping  was perfconsensus network using SplitsTree4 [In general, a topology is taken as reliable if tree reconstruction results in the same topology for at least 95% of the datasets generated by bootstrapping. This is a quite strict approach and it has been shown that subtrees of a tree may be accepted as being significant if they are supported by only 70% of the trees . Using titsTree4 . The speet al. [The program meme ,69 was uet al. . SequencAuthors CR and IH are the principle authors of this article. IH gathered the sequences and constructed and analyzed the phylogenetic trees. CR analyzed the subtype determining residues and constructed and interpreted the sequence logos. CR wrote the manuscript with the participation of IH in several sections. Authors TW and WW made important contributions to biological questions and DHH contributed to phylogenetic questions. All authors read and approved the final version of the manuscript.Phylogenetic tree of all 525 C domain sequences of this study reconstructed using phyml. Zipped Nexus file .Click here for fileLCL, Starter and DCL domain using LogoMat-P Comparison of the logos generated from the pHMMs for the 3 subtypes [ogoMat-P .Click here for filePhylogenetic tree of all 525 C domain sequences of this study reconstructed using phyml.Click here for fileHMMER outputs of glyco-NRPS: fossils in ComC and ComD. ZIP file containing two text files.Click here for fileLCL, Starter, DCL, Dual) which can be used to detect and distinguish between the subtypesProfile HMMs of the 4 complete C domain subtypes (. Zipped text file (file name extension .hmm to be used with the program package HMMER [ge HMMER ).Click here for fileAligned full length condensation domains of this study. Zipped sequence file .Click here for fileLCL, Starter, DCL, Dual)Profile HMMs of all 7 motifs of all subtypes (. Zipped text file (file name extension .hmm to be used with the program package HMMER [ge HMMER .Click here for fileListing of NRPSs from known biosynthesis clusters used in this study.Click here for fileSequence logos of all C domain motifs created with weblogo [ weblogo . ZIP filClick here for file


The reliable dissection of large proteins into structural domains represents an important issue for structural genomics/proteomics projects. To provide a practical approach to this issue, we tested the ability of neural network to identify domain linkers from the SWISSPROT database (101602 sequences).Our search detected 3009 putative domain linkers adjacent to or overlapping with domains, as defined by sequence similarity to either Protein Data Bank (PDB) or Conserved Domain Database (CDD) sequences. Among these putative linkers, 75% were "correctly" located within 20 residues of a domain terminus, and the remaining 25% were found in the middle of a domain, and probably represented failed predictions. Moreover, our neural network predicted 5124 putative domain linkers in structurally un-annotated regions without sequence similarity to PDB or CDD sequences, which suggest to the possible existence of novel structural domains. As a comparison, we performed the same analysis by identifying low-complexity regions (LCR), which are known to encode unstructured polypeptide segments, and observed that the fraction of LCRs that correlate with domain termini is similar to that of domain linkers. However, domain linkers and LCRs appeared to identify different types of domain boundary regions, as only 32% of the putative domain linkers overlapped with LCRs.Overall, our study indicates that the two methods detect independent and complementary regions, and that the combination of these methods can substantially improve the sensitivity of the domain boundary prediction. This finding should enable the identification of novel structural domains, yielding new targets for large scale protein analyses. Structural genomics/proteomics projects seek to establish high-throughput techniques by promoting routine protein structure determination either by X-ray crystallography or NMR spectroscopy -7. HowevProtein dissection is often a long and tedious process. Limited proteolysis is the prevalent experimental method for determining structural domain boundaries -12, but Various theoretical methods for identifying domains in protein sequences have recently been reported. These include well-established sequence similarity searches against existing domain databases, such as Pfam or SMART -19. A maRecently, domain prediction methods based on sequence information alone, such as the statistics of residue contact in domains , the staIn this paper, we used our neural network  to searcIn many applications, including ours, it is critical to reduce the number of false positives because of their experimental costs, while false negatives are not as detrimental. In our neural network, a 'cutoff' parameter determines the balance between specificity and sensitivity  . Thus, wFor the purposes of this discussion, we define 'putative structural domains' as sequence segments with high similarity to PDB or CDD sequences . Putative structural domains are thus able to fold into a native structure or at least to form a domain, and we used them to assess the correctness of the predicted domain boundaries. As anticipated, a substantial fraction of the SWISSPROT sequences is covered by known putative structural domains. Specifically, from a total of 101602 SWISSPROT sequences, 38470 sequences  had similarity to a PDB sequence, and 64349 sequences (43% on a residue basis) had similarity to a CDD sequence . However, the underestimations are likely to be very small, and concern only a few percents of the putative linkers, as most proteins in the PDB (and many in the CDD) are single structural domain proteins ,29.The above classification was performed by allowing an error window between the position of the predicted linker and the termini of the putative structural domain. As expected, when the error window was increased, the occurrence of correct matches increased while that of the overlaps decreased. With an error window of 20 residues, the percentages of correct matches (classes 1 and 2), overlaps (class 4) and unknown locations (class 3) were 27.5%, 9.2% and 63.4%, respectively Figure . Thus, 7Most large-scale sequence databases contain a substantial number of long, unstructured, disordered regions that may interfere with systematic searches for structural domains. Thus, the detection of unstructured portions of proteins as defined by low complexity regions (LCRs), which are unlikely to fold into a globular structure , or struAlthough both the domain linker and LCR predictions correlate well with the putative structural domain termini, it is important to note that the LCRs and linkers are located in different sequence regions. Indeed, only 2561 out of 8539 LCRs overlapped with the putative domain linkers predicted by our neural network, and, in turn, 2643 out of 8133 putative linkers were detected by the SEG program . We compared the random guesses with our neural network and SEG prediction using a quality index calculated as the ratio of correct predictions relative to the sum of correct and incorrect predictions -46, whicFrom a practical viewpoint, it is important to evaluate the error window within which the boundaries are predicted. The exact position of a domain boundary is obviously ambiguous. The first reason is that PDB sequences may include several unstructured terminal residues (without coordinates), causing some uncertainties about the exact positions of the putative structural domain termini. The uncertainty arising from the CDD sequence is even larger. Second, the smoothing windows used to reduce the spurious predictions introduce ambiguity in the positions of the predicted domain linkers, as they smear their C and N termini. These issues can be examined using an error window parameter that accommodates the positional ambiguity generated by both the putative structural domain termini and the predicted domain linkers (or LRCs). As shown in Figure Our study strongly suggests that sequence characteristics alone, as detected by either our neural network or SEG, can identify domain boundaries in protein sequences even without sequence similarity to existing domain databases. There is a clear correlation between the termini of putative structural domains and the positions of both the domain linkers and the LCRs. Furthermore, our neural network and SEG are complementary for detecting domain boundaries, and when combined, the sensitivity of the domain boundary prediction is increased without decreasing its specificity. Overall, our study shows that domain identification protocol based on domain boundary prediction can be applied to practical problems, such as the identification of novel structural domains, and thus will yield new targets for large scale protein analyses.A total of 101602 SWISSPROT protein sequences  were useWe used a two hidden units neural network  trained The smoothing window size and the threshold parameters were fixed to 19 and 0.5, respectively, as in our previous study. However, we set the cutoff parameter to values ranging from 0.90 to 0.98, because a high cutoff yields a better prediction specificity at the cost of the prediction sensitivity. The specificity and sensitivity for the first ranked domain linkers predicted with a cutoff of 0.90 are 81.8% and 10.3%, respectively, as calculated with a ten-fold jack-knife .Sequence entropy  has been used to quantify the complexity of amino acid sequences, and several studies have examined the relationship between the sequence entropy and the globularity of proteins ,43. AccoSEG is a program that identifies low-complexity regions in protein sequences . This prWe evaluated the validity of the prediction of the domain boundaries from their positions relative to the putative structural domains as defined above. The predicted domain boundaries were divided into four classes Figure , using ai.e. a prediction without any a priori information, to be the probability that a randomly assigned position matches a terminal residue of a putative structural domain. Four classes were defined similarly to those used to evaluate the putative domain linkers and the low-complexity regions. For example, a randomly picked residue was considered to be correctly located and was classified in class 1, when the end of a putative structural domain was found within the error window. The success rates  for the blind prediction, the putative domain linkers and the low-complexity regions were calculated as the rate of correct matches (classes 1 and 2) relative to both the correct and incorrect matches .We assumed the success rate of a blind prediction, S.M. designed the study, wrote the programs, analyzed the data, and wrote the paper under the supervision of Y.K. Y.K. conceived the study, analyzed the data and wrote the paper with S.M. S.Y. supervised S.M. and the study.


Protein domains are fundamental evolutionary units of protein architecture, composing proteins in a modular manner. Combinations of two or more, possibly non-adjacent, domains are thought to play specific functional roles within proteins. Indeed, while the number of potential co-occurring domain sets (CDSs) is very large, only a few of these occur in nature. Here we study the principles governing domain content of proteins, using yeast as a model species.We design a novel representation of proteins and their constituent domains as a protein-domain network. An analysis of this network reveals 99 CDSs that occur in proteins more than expected by chance. The identified CDSs are shown to preferentially include ancient domains that are conserved from bacteria or archaea. Moreover, the protein sets spanned by these combinations were found to be highly functionally coherent, significantly match known protein complexes, and enriched with protein-protein interactions. These observations serve to validate the biological significance of the identified CDSs.Our work provides a comprehensive list of co-occurring domain sets in yeast, and sheds light on their function and evolution. An overview of the identified bicliques is given in Figure A direct comparison with the combinations in Vogel et al  is not pSome of the CDSs we identified were well supported by previous studies. For example, we identified a combination consisting of the VHS domain IPR002014) and the UIM (ubiquitin interacting motif) domain (IPR003903). The VHS domain has a membrane targeting role in vesicular trafficking in eukaryotic cells 14 and th. The rolAs another example, we identified a combination of the motor region of the myosin head (IPR001609) and the IQ calmodulin-binding region (IPR000048) in the myosin family of proteins. These proteins are responsible for actin-based motility in eukaryotic cells, by using ATP hydrolysis to move on actin filaments . They arA statistically significant CDS suggests that its associated proteins are involved in similar biological processes. We examined whether the proteins in each of the CDSs exhibited functional coherency according to the gene ontology (GO) annotation (Methods). We found that 89 out of the 99 CDSs (90%) were significantly functionally coherent Figure . To avoiBicliques sharing domains or proteins were further found to relate in function, as demonstrated by the biclique network in Figure To investigate the relations between the GO terms characterizing the identified CDSs, we also created a network of GO terms, where each node is a GO slim annotation and two nodes are connected if they significantly share enriched combinations . As we had a reliability estimate to each reported interaction (Methods), we also compared the reliability distributions of within-biclique interactions and all other interactions. We found that the protein-protein interactions within bicliques were significantly more reliable than other reported interactions (p < 0.0014 by a Wilcoxon rank sum test).The functional coherence of proteins within bicliques has led us to investigate the physical connections among them. We expected proteins sharing similar CDSs to interact with one another and to match known yeast complexes. To test whether proteins sharing a particular CDS tend to interact, we compared the fraction of interacting proteins within bicliques to the overall fraction of interacting pairs. We found that proteins sharing a CDS significantly tended to co-interact  of the protein sets that had at least two MIPS annotated proteins were significantly enriched for a known complex; and 89% (8/9) of the sets having at least 3 MIPS annotated proteins were enriched.As further support for the identified functional relations between proteins sharing a CDS, we tested whether these protein sets are enriched for known protein complexes from the MIPS database . To thisancient domains, which are found also in bacteria or archaea, and new domains, which are specific to yeast , and there was an evident correlation between the score of a combination (measuring its overrepresentation) and its enrichment level (p < 0.0047 by Spearman correlation test), as demonstrated in Figure p <0.0171, by a Wilcoxon rank sum test), as further exemplified in Figure S4.Finally, we studied the age distribution of domains within CDSs. To this end, we classified the yeast domains into ast (cf. ). CDSs wIt has previously been shown that the repertoire of domain combinations in an organism's proteome is restricted to only a small fraction of the set of possible combinations . Here wesequence-based if it has a PRINTS [structure-based if it has a PDB [Our analysis relied on the Interpro database, which includes domain annotations from both structure- and sequence-based sources. In order to investigate the influence of the domain type on our results, we devised a rough classification of domains into two categories: A domain is called a PRINTS  or SMARTa PRINTS  source aas a PDB , SCOP [3as a PDB  or CATH as a PDB  source. as a PDB  source. First, we examined whether the functional coherency of bicliques is more prominent for structure- or sequence-based domains. To this end, we defined a biclique as structure-based if all its constituent domains were structure-based, and sequence-based if it contained at least one sequence-based domain.  We found that 88.5% of the sequence-based bicliques and 92.1% of the structure-based ones are functionally coherent. These rates are comparable to the observed 90% rate when considering all domains. Furthermore, 28% of the functionally coherent bicliques contained at least one sequence-based domain whose annotated function in InterPro matched the biclique's enriched function. Similarly, 70.8% of the functionally coherent bicliques contained at least one structure-based domain whose annotated function matched the enriched function. These percentages nicely match the frequencies of sequence- and structure-based domains in the IntePro domain collection.p <0.0059 for sequence-based bicliques and p <2.5e-5 for structure-based ones). We conclude that the domain type does not significantly bias the interaction enrichment results.Second, we tested the correlation between the domain type and the tendency of a containing protein to interact. Specifically, we compared the fraction of interacting proteins within either sequence- or structure-based bicliques to the overall fraction of interacting pairs. We found that proteins within bicliques of both types significantly tended to co-interact , it is S. cerevisiae. We considered only Interpro entries of type domain and family, as described in Cohen-Gihon et al. [We downloaded the Interpro  domain an et al. . In totaProtein-protein interaction data were downloaded from the DIP database  , where m represents the total number of edges in the graph [We represented the domain content of proteins using a bipartite graph of domain and protein nodes whose edges connect proteins to their constituent domains. We assigned weights to the edges reflecting the chance of observing such edges in a random graph with the same node degrees. Precisely, for an edge connecting nodes of degrees he graph .M denote the total number of proteins, let K denote the number of hubs, let N denote the number of multi-domain proteins, and let S denote the number of proteins that are both multi-domain and hub. Then the corresponding p-value is:Hub proteins were classified as in Ekman et al.  as proteP and a subset of domains D, such that each protein in P contains all the domains in D. The score of a biclique reflects the likelihood of observing such a biclique in a random, degree preserving network, and is defined as the sum of the weights assigned to the biclique's edges. We focused on maximal bicliques that contain at least two domains and at least two proteins. To detect the highest-scoring bicliques we adapted the method of Tanay et al. [7 such combinations are possible in our data). Each such combination was assigned a score according to the weight it induces with respect to the protein . Iterating over all the proteins in the data set, for each combination C we obtained the total weight of a biclique whose domain set is C. By applying the same algorithm to random protein-domain graphs with the same node degrees (see below), we were able to assign an empirical p-value to each CDS. The latter was determined by calculating the biclique's ranking in a list of 100 scores, representing the maximum weight obtained in each of 100 random runs. Only bicliques with p-value < 0.05 were retained . In total, we identified 99 significant bicliques.A biclique is defined as a fully connected bipartite subgraph, i.e., a subset of proteins y et al. ,43. BrieRandomized protein-domain bipartite graphs were created by starting with the original graph, and iteratively shuffling its edges while maintaining node degrees, using the "switch" method .p-value by comparing its hypergeometric score to that of 100 random protein sets of the same size. The resulting p-values were further corrected for multiple biclique testing using the false discovery rate (FDR) procedure. Protein sets with corrected p-values smaller than 0.05 were considered functionally coherent.Functional coherency of protein sets was based on the Gene Ontology (GO)  annotatip-values were calculated by comparing the hypergeometric scores to those obtained for random sets of proteins of the same size. These p-values were further FDR corrected for multiple testing. The fraction of sets with significant matches (p < 0.05) was measured.To quantify the correspondence between the bicliques we identified and known complexes from the MIPS database , we applFor each set of domains in a biclique, the enrichment of ancient domains was measured using a hypergeometric score and compared to the enrichments under random labelings of domains as ancient and new, respecting the size of each class.ICG, RS and RN participated in the design of the study and drafted the manuscript. ICG carried out the analyses. All authors read and approved the final manuscript


The translation initiation complex eIF3 also contains PCI- and MPN-domain proteins, but seems to deviate from the 6+2 stoichiometry. Initially, the PCI domain was defined as the region of detectable sequence similarity between the components mentioned above.The 'lid' subcomplex of the 26S proteasome and the COP9 signalosome (CSN complex) share a common architecture consisting of six subunits harbouring a so-called PCI domain  in the N-terminal region of most PCI proteins, suggesting that their homology is not restricted to the PCI domain. We also detected a previously unrecognized PCI domain in the eIF3 component eIF3k, a protein whose 3D-structure has been determined recently. By using profile-guided alignment techniques, we show that the structural elements found in eIF3k are most likely conserved in all PCI proteins, resulting in a structural model for the canonical PCI domain.Our model predicts that the homology domain PCI is not a true domain in the structural sense but rather consists of two subdomains: a C-terminal 'winged helix' domain with a key role in PCI:PCI interaction, preceded by a helical repeat region. The TPR-like repeats detected in the N-terminal region of PCI proteins most likely form an uninterrupted extension of the repeats found within the PCI domain boundaries. This model allows an interpretation of several puzzling experimental results. Mpr1-Pad1 N-terminal) [In eukaryotic organisms, there exist at least three distinct multi-protein assemblies that are jointly referred to as 'PCI complexes'  and haveerminal) ,3.Among these complexes, the proteasome lid and the CSN share a particular degree of analogy. Both complexes consist of eight core subunits, six of them of the PCI class and two of the MPN class. As described previously  and summDespite the common homology domains and a similar structure, the functions of the three PCI complexes are very different. The proteasome lid, in combination with the 'base' complex containing a hexameric ring of AAA-ATPases, forms the 19S regulatory particle, which in turn constitutes an essential subcomplex of the 26S proteasome -9. No spSo far, the metal-containing MPN subunits and the non-PCI/non-MPN portion of the complex constitute the only known carriers of functionality. The PCI proteins themselves seem to be the main building blocks of the complexes, a fact already suggested by their high abundance. There are several hints that the PCI subunits are crucial for proper complex assembly -19. The During an exhaustive bioinformatical analysis of proteasome subunits and other components of the ubiquitin/proteasome system, we obtained two independent results jointly suggesting that a structure-based redefinition of the PCI domain is appropriate: on one hand, we detected multiple instances of TPR-like repeats in the N-terminus of many PCI proteins, which suggests that the homology between the proteasome and CSN components is not restricted to the PCI domain itself. On the other hand, we detected a previously overlooked PCI domain in the novel eIF3 subunit eIF3k . Most inS. bayanus . The TPR repeat family [In most PCI proteins, the canonical PCI domain occupies a region of approximately 190 residues close to the carboxy-terminus of the sequence. The N-terminal non-PCI portion of the proteins is moderately conserved between species and only poorly conserved between different PCI subunits – even between the analogous subunits of the lid and the CSN. Upon submitting those PCI proteins to profile- or HMM-based domain detection services, no significant matches were obtained for the N-termini of the proteins. However, the PROSITE  profile .01, Ref ) and Csnt family  is very To further investigate if a TPR-like structure should be assumed for the N-terminal portions of all PCI proteins, we performed a secondary structure prediction for each of the protein families individually. To that aim, we constructed multiple alignments for representative members of each subunit family and submitted the alignment to PHD and JPred prediction servers ,31. As aThe predicted all-helical secondary structure of the non-PCI portion of lid and CSN subunits consists of several short helices that appear to occur in pairs. To test whether those bi-helical segments correspond to the structural elements of a TPR-like repeat, we selected several examples starting immediately upstream of the predicted PCI domains. When multiple alignments of those bi-helical segments were used for profile construction and in subsequent database searches several bona fide TPR proteins were found to match within the TPR region, with the bi-helices being in the correct TPR register, these segments were also classified as TPR-like. No matches to established HEAT- or Armadillo-repeat proteins were found, demonstrating that the scores are not just caused by an arbitrary helical repeat arrangement.It should be pointed out that none of the singular observations described above is able to prove a statistically significant sequence relationship between the N-terminal portions of PCI proteins and true TPR-repeats. Taken as a whole, the results strongly suggest that there is a general tendency of PCI domains to be preceded by an α-helical repeat structure that has at least some specific relationship to the tetratrico-peptide repeat.In the first surveys of recognizable PCI domains, only three PCI subunits of the eIF3 complex had been detected . More reA generalized profile was constructed from the conserved portion of representative eIF3k orthologs from vertebrates, invertebrates, plants and fungi. After a scaling step, the resulting profile was run against a nonredundant protein database. Apart from the eIF3k proteins already used for profile construction, the only other sequences matching with significance were selected PCI subunits of the proteasome and the CSN, among them rice Csn8 p = 0.01) and the drosophila Rpn12 homologue (p = 0.05). All of the twenty top-scoring sequences could be identified as either Csn8- or Rpn12-homologs. As shown in table  and the The discovery of a PCI domain in eIF3k is of particular importance, as a three-dimensional structure of eIF3 has been solved recently . So far,A detailed analysis of the eIF3k structure  reveals 10 helices that connect the helical hairpins in eIF3k are absent in most other PCI proteins. As mentioned in the previous paragraphs, there are several instances of subsignificant sequence similarity to TPR repeats found also within the N-terminal subdomain of the PCI domain. By contrast, no similarity to HEAT or Armadillo-repeats has been observed. Thus, we prefer to interpret the helical hairpin structure of the N-terminal subdomain as atypical TPR-like repeats rather than as the HEAT/Armadillo repeats suggested by Wei et al. [Within the N-terminal subdomain, the sequence conservation between the different PCI domain families is relatively poor and some aspects of the alignment shown in figure i et al. .The globular C-terminal subdomain (WH) is generally better conserved than the helical N-terminal domain and as a consequence, the part of the alignment covering this structural subdomain shown in figure Taken together, the comparison of the PCI alignment with the eIF3k structure , eIF3c (EIF3S8) and eIF3e (EIF3S6) . Recent Comparing the stoichiometry of eIF3 with the two better-conserved PCI complexes, only one PCI subunit seems to be missing. Our sequence analysis efforts have also included other known eIF3 subunits, but no indications for further PCI domains could be obtained (data not shown). Given the high degree of PCI sequence divergence, it cannot be fully excluded that one of the non-PCI/non-MPN subunits  harbours a cryptic PCI domain that has eluded our detection. On the other hand, it is well conceivable that eIF3 has a deviating subunit composition. In yeast and several other organisms, not only the MPN proteins are missing but also the number of PCI components is reduced, as eIF3e and eIF3l are absent. At present, it is not clear whether the corresponding positions in the complex are left empty or are filled by additional copies of the remaining PCI components. In evolutionary terms, it appears likely that the eIF3 complex is a 'degraded' copy of an ancient lid-like complex, which has lost its MPN+/JAMM mediated catalytic activity and potentially some of its PCI subunits. In turn, by acquiring a group of novel non-PCI/non-MPN subunits, the eIF3 complex has gained a functionality that is different  to the proteasome lid and its cousin, the CSN complex.In our original discovery note , we had Using the eIF3k-derived structural model, most of these observations can be readily explained. The C-terminal PCI/PINT boundary, which is agreed on by all domain databases, corresponds to the C-terminal boundary of the structural WH-like domain. The N-terminal boundary of the PINT domain, as described in the SMART database, essentially corresponds to the N-terminus of the WH-like domain. The PCI domain of the Pfam database corresponds to the WH-portion plus a single α-helical hairpin repeat. Finally, the PCI domain as described in the PROSITE database covers the WH-portion and all three helical hairpin repeats found in the eIF3k structure. Of the three representations, the PINT domain of the SMART database is structurally most correct, as it describes a true autonomously folding domain. The observation that some PCI families lose their sequence conservation at different N-terminal positions can be explained by assuming a variable number of helical repeat motifs for those proteins. As an extreme example, only the WH-like region could be detected in eIF3e by our profile searches, and the secondary structure prediction for the eIF3e family suggests a β-structure instead of the usual helical-hairpin repeats upstream of the WH region. This finding can be taken as a further hint for structural and functional independence of the N- and C-terminal sub-regions of the PCI homology domain.PCI-associated module) with TPR-like properties has been proposed [Our finding of TPR-like repeats preceding many PCI domains, combined with the helical repeat structure of the N-terminal portion of the PCI domain itself, leads to the interesting question if these repeats are of the same type and may form a continuous solenoid structure. The authors of the eIF3k crystal structure propose a structural relationship between the eIF3k N-terminus and the HEAT motif based on superposition calculations with DALI . By contproposed . Our finDue to the borderline sequence similarity between the classical TPR motif and the distinct helical hairpins of the PCI proteins, a completely novel type of bi-helical repeats distinct from TPR and HEAT/Armadillo or some kind of intermediary form cannot be ruled out. Structurally, HEAT and TPR repeats are relatively similar and both tend to form superhelical solenoid structures . WithoutPCI proteins constitute the main components of the proteasome lid and the CSN complex and also form the structural core of the translation initiation factor eIF3. So far, no catalytic activity has been described for PCI proteins. Given the lack of invariant polar residues, such a role appears unlikely. The role of the PCI domains is most likely that of a scaffold for the other complex subunits and other binding partners. There are at least three distinct structural roles that PCI proteins have to fulfil: i) maintaining the integrity of the complex by binding to other PCI proteins, ii) attaching the MPN-subunits to the complex, and iii) binding to other partners such as the base-complex in the case of the proteasome lid or the RNA-binding subunits of the eIF3 complex.The assignment of these functionalities to the different regions of the PCI proteins, and equally important, the source for the subunit interaction specificity or promiscuity have been subject to several experimental studies, some of them published while others have been presented at a recent meeting on PCI complexes . The PCITsuge et al. analysed truncated forms of human Csn1 for its interaction with other PCI subunits of the CSN complex . A constIn summary, we believe the PCI domain could play a role as a universal binding domain supporting intra-complex interactions as well as recruitment of additional ligands. The model presented here is a first step to the understanding of the supramolecular architecture of three important complexes and certainly will facilitate the interpretation of further experimental results. Nevertheless, a full understanding of the interaction mode between PCI- and MPN-domain proteins will certainly require experimentally determined high-resolution structures of the components – or ideally, that of an intact complex.pftools package, version 2.1. (program available from the URL ). Generalized profiles were constructed using the BLOSUM45 substitution matrix [Sequence database searches were performed with a nonredundant data set constructed from current releases of SwissProt, TrEMBL, and GenPept ,41. Genen matrix  and defan matrix . DatabasFor sufficiently related proteins, multiple alignments were calculated by T-coffee , using eFor each subfamily-specific multiple alignment, secondary structure elements were predicted using web services of PHD  and JPreHS performed the sequence analysis and participated in writing the manuscript. KH designed the study, participated in the sequence analysis and in writing the manuscript. All authors read and approved the final manuscript.


The mosaic sperm protein zonadhesin (ZAN) has been characterized in mammals and is implicated in species-specific egg-sperm binding interactions. The genomic structure and testes-specific expression of zonadhesin is known for many mammalian species. All zonadhesin genes characterized to date consist of meprin A5 antigen receptor tyrosine phosphatase mu (MAM) domains, mucin tandem repeats, and von Willebrand (VWD) adhesion domains. Here we investigate the genomic structure and expression of zonadhesin-like genes in three species of fish.zlg) in Atlantic salmon  were sequenced. Zlg is similar in adhesion domain content to mammalian zonadhesin; however, the domain order is altered. Analysis of puffer fish (Takifugu rubripes) and zebrafish (Danio rerio) sequence data identified zonadhesin (zan) genes that share the same domain order, content, and a conserved syntenic relationship with mammalian zonadhesin. A zonadhesin-like gene in D. rerio was also identified. Unlike mammalian zonadhesin, D. rerio zan and S. salar zlg were expressed in the gut and not in the testes.The cDNA and corresponding genomic locus of a zonadhesin-like gene (T. rubripes and D. rerio and uncovered zonadhesin-like genes in S. salar and D. rerio. Each of these genes contains MAM, mucin, and VWD domains. While these domains are associated with several proteins that show prominent gut expression, their combination is unique to zonadhesin and zonadhesin-like genes in vertebrates. The expression patterns of fish zonadhesin and zonadhesin-like genes suggest that the reproductive role of zonadhesin evolved later in the mammalian lineage.We characterized likely orthologs of zonadhesin in both  Sus scrofa) ZAN was first described by Hardy et al.  was sequenced by primer walking from both directions and assembled to give a 4388 bp sequence. PCR primers were designed to the 3'-end of the EST and used to amplify a probe for hybridization to Atlantic salmon genomic DNA and bacterial artificial chromosome (BAC) library filters  analysis of Atlantic salmon. We assembled this zonadhesin-like gene  signal of AATAAA was identified by Genscan . The ZV3 assembly contained only one copy of zonadhesin that also assembles into linkage group 7. It is important to note that the ZV3 assembly consisted entirely of whole genome shotgun reads; the ZV4 assembly incorporated finished BAC sequence into the ZV3 assembly; and that both assemblies are considered 'pre-assemblies' that need to be analyzed with caution. The location of zonadhesin at linkage group 7 is supported by the fact that other genes with significant similarity to human chromosome 7q22 map to the same region of the D.rerio genome. These genes include acetylcholine esterase, serpine, AP1S1, unnamed product FLJ39237, unnamed protein product FLJ10925 and mucin  that aligned to a 600 kb region surrounding the zan locus at linkage group 7, or a 1 MB region at linkage group 24, were obtained and aligned to both loci using the BLASTZ algorithm through the MultiPipMaker web server. Our analysis showed that the zonadhesin-containing BAC BX649275 integrated completely into linkage group 7 and was ≅ 97% identical overall, not including indels. Seven additional BACs from the DKey library aligned to the scaffold and produced an acceptable tile through the entire zan locus . The 4,616 amino acid translation product of the putative 9275. Thezan locus at linkage group 7 only contains two of these ≈ 5 kb repeats. It is possible the third repeat was collapsed in the whole genome shotgun assembly process or the presence of two repetitive blocks is a true population variant.Zebrafish zonadhesin has expanded its VWD domains through recent tandem duplication. The VWD domains 5, 6 and a fragment of 7 are encoded in 17 exons (45–61). These exons have identical sizes to the exons (11–27 and 28–44) encoding the first four VWD domains. Furthermore, each group of 17 exons are symmetrical and flanked by phase '1' introns, which is evidence for recent domain expansion. Pair-wise alignment of BAC BX649275 against itself revealed these exons are found in three ≈ 5 kb blocks that are 85–87% identical. The zan we extracted gut and testes RNA and performed RT-PCR and Northern blot analysis. Three different PCR primer sets were designed against three regions of the predicted zan cDNA. The first primer set was designed to amplify the exons encoding the MAM domains through to the first VWD domain. This primer set produced a doublet in both male and female   Figure . The fem) Figure . These r) Figure  and 5C. ache gene. This syntenic relationship was also found at the mammalian and zebrafish zan loci  revealei Figure . HoweverPuffer fish zonadhesin was predicted to contain 47 exons that coded for a protein of 2,525 amino acids. The predicted zonadhesin protein contained two MAM domains at the N-terminus, a mucin domain, five VWD domains, an EGF domain, a transmembrane domain and a short cytoplasmic domain  genome projects. This search uncovered several regions with VWD-containing proteins without any detectable MAM domains, some of these possibly related to FCGBP proteins. One interesting exception was a zonadhesin-like gene found on zebrafish chromosome 2. This putative zebrafish zlg encodes a 1,308 amino acid protein containing three MAM domains, the first of which is flanked by short (15 amino acid) mucin-like low complexity regions. The MAM domains are followed by two VWD domains. This gene structure is reminiscent of S. salar Zlg and shows that a zonadhesin ortholog and a zan-like gene exist together in the zebrafish genome  did not reveal an obvious zonadhesin ortholog but rather a prediction of a FCGBP-like protein residing on chromosome 9 [GenBank: XP422715.1]. We analysed the corresponding region of the G. gallus genome and utilized additional EST evidence and in silico predictions to obtain a putative transcript that encodes a 4,770 amino acid gene product. This gene product contains three MAM domains in addition to VWD and mucin-type O-glycosylation sites after each of the MAM domains. Overall, this MAM-mucin-TIL-VWD series of domains is reminiscent of all zonadhesins and is evidence for a common evolutionary origin of zonadhesin, the zonadhesin-like genes and the Fc fragment of IgG binding protein(FCGBP).The search for zonadhesin-like genes in chicken , suggest that zonadhesin was also 'hijacked' by the mammalian reproductive system.Many mammalian zona pellucida adhesion molecule candidates appear to have evolved from different physiological processes. Well known examples of sperm proteins with enzymatic function that have been 'hijacked' into playing non-enzymatic roles in sperm-egg interactions include: B4GALT1/GalTase , SPAM1/PH-20 , HK1/ZRK/p95 (hexokinase), and ARSA/SLIP1 . Evidenzan was expressed in the gut but not in the testes. In addition to these orthologs, we characterized zonadhesin-like genes (zlg) in Atlantic salmon, zebrafish and chicken. While the Atlantic salmon zlg contained the same domains found in zonadhesin, the order of these domains was altered and the expression was found predominantly in the gut and not in the testes. Overall, this suggests that zonadhesin's reproductive role evolved later in the mammalian lineage.We identified zonadhesin genes in zebrafish and puffer fish that are similar in domain order and content to all known mammalian orthologs. Unlike all mammalian zonadhesin genes studied to date, zebrafish CK990464]. These five filters contained 91,776 BAC clones in a pTARBAC2.1 vector with an average size of 190 Kb. Each filter was estimated to represent the salmon genome once. Filter hybridizations were conducted as described by CHORI . Primers were designed to the predicted translation start site on the genomic DNA in order to amplify fragments spanning the 5' end of the coding region from gut total cDNA. Sim4  and 1544 bp [GenBank: AF029250].RNA was extracted from Atlantic salmon tissues  and from zebrafish testes and gut using Trizol (Invitrogen). Total RNA samples were quantified and checked for quality by spectrophotometric analysis and agarose gel electrophoresis. Northern blots were prepared using the NorthernMax-Gly kit (Ambion) following the manufacturer's instructions. Ten μg of Atlantic salmon or 5 μg of zebrafish total RNA from each tissue was blotted on a Hybond positively charged nylon membrane (Amersham). Northern blot analysis of Atlantic salmon tissues utilized the same probe 1 described for the Southern blot analysis. The zebrafish zonadhesin probe was amplified from gut tissue using a primer set spanning from the 3' end of the epidermal growth factor domain through to the cytoplasmic domain using primers 5'-GGTTTGAGGGCACAAACTGT-3'and 5'-TAGGGATGCGCTGTCTTTTT-3'. Prehybridization for both Northern blots proceeded for 2 h at 42°C in 15 mL of ULTRAhyb buffer (Ambion). Hybridization with the αzlg probe 1 primers or ubiquitin primers (5'-ATGTCAAGGCCAAGATCCAG-3' and 5'-TAATGCCTCCACGAAGACG-3'). The zlg EST [GenBank: CK990464] and the 722P12 BAC were included as positive controls and both primer sets were run with template-free negative controls. For zebrafish, three primer sets were designed against the genomic zan sequence. The first primer set flanked the second MAM domain through to the first VWD domain (5'-TTGCAATTGATAGCGTCTGC-3' and 5'-TTCAGTCACAGGGTCACAGG-3'); the second primer set flanked the ninth VWD domain (5'-GGAGACCGTTACTGCAAACC-3' and 5'-CGAACAGTGATGCCGTACAC-3'); and the third primer set stretched from the epidermal growth factor domain to the cytoplasmic domain (see Northern blot probe description). The integrity of each cDNA was confirmed by control PCR reactions that used an ubiquitin primer set (5'-CCTCGAGGTAGAGCCAAGTG-3' and 5'-GCAGCACACAAGGTGCAAAGTA-3') and a template-free negative control.Total RNA from Atlantic salmon and zebrafish tissue extracted as above was reverse transcribed using Superscript II enzyme (Invitrogen) and an 18 nucleotide oligo d(T) primer as described in the manufacturer's protocol with exception of the production of the cDNA template for the zebrafish MAM primer set which required a gene-specific internal primer for reverse transcription to reach this region (5'-AGACACTTTCACCCCCAGTG-3'). For Atlantic salmon, one μL of cDNA was amplified in a 25 μL reaction volume with either zan nucleotide sequences. Scaffold 870 from assembly 2 was found to have similarity to zonadhesin and was subsequently analyzed by Genscan for coding sequences and peptide predictions. Puffer fish ESTs were aligned to scaffold 870 using Sim4.The puffer fish zonadhesin was found by BLASTN search of the three puffer fish genome assembles available at the MRC RFCGR Fugu genome database  with humThe zebrafish zonadhesin was found by BLASTP search of the Ensembl zebrafish peptide database (Ensembl assembly 25.4.1) using a fragment of Atlantic salmon predicted protein as the query sequence. The Atlantic salmon query fragment consisted of all the amino acids except those representing the MAM domains. The two genomic regions identified were analyzed by Genscan to find the putative coding and protein sequences.Gallus gallus) genome projects using the ENSEMBL BLAST search tools using both cDNA and protein sequences from several zonadhesin-like genes as in silico probes. These included salmon zlg, human, puffer fish and zebrafish zans and the related human FCGBP gene. Genomic regions from all significant matches were extracted and gene prediction analysis was performed using Genscan.We looked for zonadhesin-like genes in puffer fish, zebrafish and chicken  of VWD domains used to construct the phylogeny in Figure Click here for fileMultiple sequence alignment (Fasta format) of MAM domains used to construct the phylogeny in Figure Click here for file