INTRODUCTION

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The functions of only a small fraction of known proteins have been determined by experiment. As a result, the use of computational sequence analysis tools is essential for the annotation of novel genes or genomes, and the prediction of protein structure and function. Currently, the most informative of these techniques are database search tools such as BLAST (1) and FASTA (2) that identify similar sequences with associated statistical significance estimates. Current limitations of the use of these programs concern less the aspects of search sensitivity and more the functional annotation of identified homologues. Annotation terms such as "hypothetical protein" or "suppressor of spt3 mutations" are helpful neither to the user's prediction of structure and function, nor to computational procedures attempting to automatically predict function from sequence.

An additional aspect concerns the annotation of complete genomes. Existing eubacterial and archaeal genomes have been analyzed with little regard to the existence of domains, because multidomain proteins in these organisms are relatively few in number. The domain as a functional and structural unit in eukaryotic proteins, however, is pre-eminent. For example, the majority of human extracellular proteins are multidomain in character (for reviews see refs. 3 and 4) and many complex eukaryotic signaling networks involve proteins containing multiple domains with catalytic, adaptor, effector, and/or stimulator functions (5). Several dozen of such "signaling domains" are known (for a review see ref. 6). The importance of modular proteins in disease is emphasized by the recent observation that the majority of positionally cloned human disease genes encode multidomain proteins, many of which are, in fact, signaling proteins (7). On the other hand, the view of the domain as a fundamental unit of structure and function is not universally accepted: not a single noncatalytic signaling domain is annotated in the widely distributed Saccharomyces cerevisiae genome directory that catalogs the genes of this complete genome (8).

Thus, there is a need to coordinate knowledge stored in the literature with that stored in sequence databases to facilitate the research of those in the scientific community who require the annotation of genes and genomes. It is our goal to provide an extensively annotated collection of cytoplasmic signaling domain alignments that enables rapid and sensitive detection of additional domain homologues as a Web-based tool.

Because it is difficult to distinguish those domains that perform cytoplasmic signaling roles from those that primarily function in transport, protein sorting, or cell cycle regulation, and for reasons of brevity, we shall discuss those domains that fall under two categories. (i) Cytoplasmic domains that possess kinase, phosphatase, ubiquitin ligase, or phospholipase enzymatic activities or those that stimulate GTPase-activation or guanine nucleotide exchange; these activities are known to mediate transduction of an extracellular signal toward the nucleus resulting in the initiation of a cellular response. (ii) Cytoplasmic domains that occur in at least two proteins with different domain organizations, of which one also contains a domain that is categorized under 1) (for a complete list of such domains see Table 1).

Domain collections that cover a wide spectrum of cellular functions do exist in the forms of motif, alignment block, or profile databases such as PROSITE (9), BLOCKS (10), PRINTS (11), or Pfam (12) and provide a guide for the annotation of new proteins. However, there is a necessary trade-off in these collections between exhaustive coverage of domains and optimal sensitivity, specificity, and annotation quality. We have chosen to initiate the collection of gapped alignments of signaling domains because these are imperfectly covered in large collections and often include homologues with extremely divergent sequences. This collection is designed to be updated easily and is provided with a Worldwide Web interface enabling automatic sequence annotation with evolutionary, functional, and structural information. The resulting SMART procedure, a simple modular architecture research tool, offers a high level of sensitivity and specificity coupled with ease of use.

	METHODS

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Construction of Multiple Sequence Alignments and Choice of the Search Program. Of the 86 domain families, multiple alignments of 83 had been published previously (for references, see the annotation that accompanies the SMART Web site). These alignments were refined according to constraints described elsewhere (13) that included minimization of insertions/deletions in conserved alignment blocks, optimization of amino acid property conservation within these blocks, and closing of unnecessary gaps within insertion/deletion regions. Gapped alignments were constructed in preference to ungapped ones to allow the prediction of domain limits and as a result of their greater information content. Care was taken to build alignments that encompassed all secondary structures of domains whose tertiary structures are known. For remaining domains, investigations of sequence similarities beyond previously published domain limits were undertaken; this resulted in N-terminal extension of the previously described PX domain alignment by a single predicted -strand, and identification of a conserved N-terminal motif in guanine nucleotide exchange factors for Ras-like GTPases. Prediction of domain limits also was aided by close proximities of domains to others with well-known limits, and to bona fide N- and C-terminal residues.

Assignment and Calibration of Thresholds for Automatic Runs. Score thresholds are required to provide automatic assignment of true positives and true negatives. There is no current method, including those that provide E- or p-value representations of score significances, that may be relied on to provide reliable values for these thresholds in all cases. As a result, manual intervention was necessary to estimate threshold values on the basis of published homology arguments and, for example, on the results of individual BLAST or Ssearch queries. SWise (16) was chosen as an established algorithm able to provide similarity scores for query sequences when compared with the alignment database; however, the SMART database method can be applied to any algorithm that provides similarity scores.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Calibration of thresholds. Selection of thresholds from the distributions of SH3 domain scores. (Upper) A histogram of SWise scores for the best match (optimal alignment; in green) of proteins with a SH3 domain profile. (Lower) Similar histograms for the second- and third-best matches (suboptimal alignments; in light blue and dark blue, respectively). Optimal alignment scores less than threshold Tp are mostly derived from sequences considered unlikely to contain SH3 domain homologues. Threshold Tp was selected as the lowest scoring true positive. Domains that are repeated twice or more in the same protein that each score above a lower threshold (Tr) are considered to be true negatives.

Seeding and Updating Procedure. To reduce redundancy and subfamily bias within sequence families, seed alignments were calculated by using an iterative semiautomatic procedure. In a first step all database sequences considered homologous, given the threshold procedures described above, are subjected to a CLUSTALW phylogenetic tree construction (24). Only a single sequence from every branch of the tree that is shorter than a defined threshold (the default distance is 0.2, which corresponds approximately to 80% identity, ref. 24) is retained in the alignment. From this seed alignment, a profile is derived leading to reiteration of the database search procedure until convergence. For example, four iterations were required to build a Src homology 2 (SH2) seed alignment containing 95 sequences, of a total of 548 SH2 domains identified in the translated EMBL sequence database.

Implementation into a Web Server. SMART has been provided with a user interface (http://www.bork.embl-heidelberg.de/Modules/sinput.shtml) that allows rapid and automatic annotation of the signaling domain composition of any query protein sequence. A graphical display is provided showing domain positions within the query sequence. The SMART set of signaling domains is annotated extensively via hyperlinks to Medline and the Molecular Modeling Database via Entrez (25), thus providing easy access to information relating sequence, homology, structure, and function. As the set of signaling sequences is necessarily incomplete and as there may be other domains represented in the query sequence, direct access also is provided to Pfam (12), a domain database that includes a variety of different domain types, yet provides a lower representation of signaling domains and with lower sensitivity (see Discussion). Intrinsic features of the query such as coiled coil regions (26), low complexity regions (27), and transmembrane regions (28) also are displayed. Annotated or unannotated regions of the query sequence are able to be subjected individually to gapped BLAST searches (1), thus allowing the advantage of a reduced search space enabling higher sensitivity in searches.

Benchmarking Protocol. To assess the sensitivity and selectivity of SMART, results were compared with annotations held by SwissProt, because this represents the best-annotated protein sequence database extant, (and includes all those annotations covered by the PROSITE database) as well as with the Pfam domain collection, because this represents the most comprehensive set of gapped alignments available (12). Our intention here was not to provide justifications for the inclusion or exclusion of particular sequences in domain alignments, but to compare literature information as represented by the SMART database, with the same information as represented by SwissProt and Pfam databases. All S. cerevisiae and human sequences were extracted from SwissProt and annotated by using the SMART protocol. Because these organisms are well-studied and their proteins relatively well-annotated they represent a stringent test for annotation procedures. The SMART domain annotations were compared manually with those derived from HMMer (14) analysis, and those contained in SwissProt (Table 1); the hmmfs program and a 25-bits threshold was used for the HMMer analysis. As the SwissProt release 34 does not contain all yeast sequences, the complete set of S. cerevisiae ORFs also was subjected to SMART analysis (Table 1).

	RESULTS

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Comparison with SwissProt and Pfam. Of all protein sequence databases, SwissProt is the most extensively annotated, making use of literature- and sequence-derived (9) data as source material. As a result the SwissProt database is a valuable resource for investigators searching for hints of the structure and function of their sequences of interest. Consequently, it is appropriate to compare SMART-derived annotations with those contained in SwissProt.

Identification of Signaling Domains in Yeast. Annotation of the complete yeast genome (6218 ORFs) revealed that 420 yeast proteins (6.7%) contain at least one of the domains included in SMART. This is larger than a previous estimate that 2% of yeast proteins are involved in signaling (8), which approximates to the percentage of S. cerevisiae proteins known to be kinase homologues. SMART identifies a total of 622 domains (Table 1); two or more domains occur in 96 of the 420 signaling proteins. Results of the SMART annotation of yeast proteins identified are summarized in a Web page (http://www.bork.embl-heidelberg.de/Modules/syeast.html), which was generated by using SMART's graphical output features.

Domain Annotation and Deduction of Functional Features. During construction of the SMART database, tensin and focal adhesion kinase (pp125^FAK), which both are localized to focal contacts, were found to contain previously unrecognized domains. Fig. 2a shows the modular architecture of tensin, an actin filament capping protein that is known to contain large coiled coil regions, an SH2 (29) and an N-terminal domain homologous to protein tyrosine phosphatases (PTPs) (20). SMART predicts a phosphotyrosine binding domain (PTB; also called phosphotyrosine interaction [PI] domain) (Table 1) in tensin's most C-terminal region, which has not previously been ascribed a domain homology. Each of tensin's three globular domainsPTP, SH2, and PTB/PIhave been implicated in phosphotyrosine-mediated signaling. This is consistent with previous findings that tensin is a substrate of the tyrosine kinase pp125^FAK (30), which is also highly tyrosine-phosphorylated when activated (reviewed in ref. 31).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Schematic representations, produced using SMART, of the domain architectures of proteins discussed in the text. See Table 1 for the identified domains; gray lines (no SMART match) might contain other known domains not included in SMART. Putative homologues were identified during SWise (16) searches and/or PSI-BLAST (1) searches (E < 0.01). (a) Domain recognition: A novel PTB domain was identified in tensin, resulting in completion of its modular architecture assignment. A PSI-BLAST search with a previously predicted PTB domain in C. elegans F56D2.1 (53) yields the tensin PTB after four passes. Prediction of molecular function via domain hit: Identification of a domain homologous to band 4.1 protein in focal adhesion kinase (FAK) isoforms. FAKs are predicted to bind cytoplasmic portions of integrins in a similar manner to that of talin, another band 4.1 domain-containing protein. A PSI-BLAST search with a band 4.1-like domain (41  HUMAN, residues 206-401) revealed band 4.1-like domains in human, bovine, and Xenopus FAK isoforms by pass 3. (b) Detection of new domains because of search space reduction: Putative DEP domains in ROM1 and ROM2 were identified by using SWise (16) and HMMer (14), but could not be detected by using PSI-BLAST. Analysis of the regions surrounding identified domains revealed the presence of a novel domain in the C-terminal regions of ROM1 and ROM2 that occurs also in several Ste20-like protein kinases, and mouse citron (CNH, citron homology). A gapped BLAST search of the region of citron C-terminal to its PH domain (CTRO  MOUSE, residues 1134-1457) reveals significant similarity with yeast ROM2 (E = 1 × 105). (c) Functional predictions for an entire domain family: A region of p62 known to bind ubiquitin (40), and its homologous sequence in the Drosophila protein ref(2)P, scored as the highest putative true negatives in a SWise search. We predict ubiquitin-binding functions for UBA domains. PSI-BLAST searches were unable to corroborate this prediction. (d) Prediction of cellular functions: Although not indicated in the primary sources (43, 44), a DEATH domain was found in rcm and other UNC5 homologues, in agreement with a previous claim (41). At the molecular level, this domain in UNC5 is predicted to form a heterotypic dimer with an homologous domain in UNC44 implying a cellular role in axon guidance. A gapped BLAST search with the known DEATH domain of death-associated protein kinase (DAPK  HUMAN, residues 1304-1396) predicts a DEATH domain in rat UNC5H1 with E = 9 × 103). (e) Signaling domains in "disease genes": Pyrin or marenostrin, a protein that is mutated in patients with Mediterranean fever and is similar to butyrophilin, contains a SPRY domain. PSI-BLAST with the SPRY domain of human DDX1 (EMBL:X70649, residues 124-240) yields a butyrophilin homologue by pass 5 and pyrin/marenostrin (residues 663-759) by pass 7. (f) Homologues of domains involved in eukaryotic signaling may not be eukaryotic-specific: DAG kinases have been found previously in mammals, invertebrates, plants, and slime mold. However, it is apparent that DAG kinase homologues of unknown function are present in yeasts and in eubacteria (see Fig. 3). A gapped BLAST search with Bacillus subtilis bmrU (BMRU  BACSU) yields significant similarities with Arabidopsis thaliana DAG kinase (KDG1  ARATH; E = 4 × 104) and a Schizosaccharomyces pombe ORF (SPAC4A8.07c; E = 1 × 107). (g) Identification of potential misclassifications: A PH domain and the lack of an obvious transmembrane sequence indicates a cytoplasmic and signaling role for a protein (INT1  CANAL) previously thought to be a yeast integrin. A PSI-BLAST search with the N-terminal PH domain of pleckstrin yielded INT1  CANAL in pass 3.

Reducing the Search Space Enables Identification of Novel Domains. S. cerevisiae ROM1 and ROM2 are sequence-similar proteins that each contain a PH domain and a RhoGEF domain that stimulates exchange of Rho1^GDP with Rho1^GTP (34). Construction of the SMART databases led to the identification of a putative DEP domain (35) in both ROM1 and ROM2 (Fig. 2b). Comparison of the ROM1 and ROM2 sequences showed a further region of similarity C-terminal to their PH domains. This region ["citron-homology" (CNH) domain] was identified as being homologous to the mouse Rho^GTP/Rac^GTP-binding protein, citron (36) and to the C-terminal regions of several Ste20-like protein kinases (Fig. 2b). A novel domain family (VHS) of unknown function(s) also has been detected in Vps27, Hrs, and STAM, and other proteins.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Multiple alignments of selected RasGEFN domains. A conserved region was found in the N-terminal regions of several proteins with RasGEF (Cdc25-like) domains (37). Surprisingly, this N-terminal domain may be present in the sequence either close to, of far from, the RasGEF domain. A PSI-BLAST search using a region (residues 898-946) of C. albicans Cdc25 (CC25  CANAL) and E < 0.01, identified each of the sequences in Fig. 3 within nine passes before convergence. Predicted (54) secondary structure and 90% consensus sequences are shown beneath the alignments; SwissProt/PIR/EMBL accession codes and residue limits are given after the alignments. Residues are colored according to the consensus sequence [green: hydrophobic (h), ACFGHIKLMRTVWY; blue: polar (p), CDEHKNQRST; red: small (s), ACDGNPSTV; red: tiny (u), AGS; cyan: turn-like (t), ACDEGHKNQRST; green: aliphatic (l), ILV; and, magenta: alcohol (o), ST). The SwissProt sequence KMHC  DICDI has been altered to account for probable frameshifts.

Deducing Functional Features of a Domain Family Via a Protein Hit. Although rare, we have identified additional members of a domain family in regions of proteins that already have been shown to perform particular functions. Such findings often suggest comparable functions for all other members of the domain family. The ubiquitin-associated (UBA) domain (Table 1) has been shown to be contained in several enzymes implicated in ubiquitination (39). We have identified a UBA domain in a region of p62, a phosphotyrosine-independent ligand of the p56^lck SH2 domain (40) that is known to bind ubiquitin (Fig. 2c). Ubiquitin-binding functions are predicted for other UBA domains.

Prediction of Cellular Function. Particular domains have been implicated in certain cellular events. For example, DEATH domains (Table 1) are present in proteins associated with apoptosis and/or axonal guidance (41, 42). Recent reports (43, 44) identify the rostral cerebellar malformation gene product (rcm) and similar homologues as putative netrin receptors. These reports do not indicate the presence of a DEATH domain in rcm or its homologues, even though the domain's presence may be readily demonstrated by sequence analysis (Fig. 2d) or from its identification in the rcm Caenorhabditis elegans orthologue, UNC-5 (41). As the DEATH domain of UNC-5 is not annotated in databases, this is one of many instances where the potential of domain identification to predict cellular function has been unfulfilled. DEATH domains often form homotypic or heterotypic dimers (42). Because DEATH domain-containing proteins UNC-44 (45) and the putative netrin-receptor UNC-5 are known to be involved in axonal guidance, we predict that transduction of the netrin-initiated signal involves heterodimerization of UNC-5 and UNC-44 DEATH domains.

Identification of Signaling Domains in Genes That Are Involved in Diseases. A recent study of 70 positionally cloned human genes mutated in diseases found that a significantly high proportion of these "disease genes" possess roles in cell signaling (7). In accordance with this, the SMART alignment database contains several novel signaling domains in these genes (including the DEATH domain in rcm-like netrin receptors, see above). Fig. 2e shows the modular architecture of pyrin (46) (also called marenostrin; ref. 47). Mutations in the pyrin gene result in Mediterranean fever syndromes that are inherited inflammatory disorders. In addition to its ret-like zinc finger, pyrin/marenostrin and other butyrophilin-like homologues contain a SPRY domain, a domain of unknown function found triplicated in ryanodine receptors and singly in other proteins (48) (Table 1). Midline 1, a pyrin-homologue that also contains a SPRY domain, is mutated in patients with Opitz G/BBB syndrome (49).

Identification of Domains in Different Phyla. The range of species in which a particular domain type is found can correlate with the evolution of specific signaling pathways; many of the known cascades are expected only in animals or eukaryotes (3). Thus, identification of DAG kinase homologues in yeast and eubacteria (Fig. 2f) is clearly a surprise. Although further experimentation is required to infer functional features, the presence of conserved, presumably catalytic, residues in the alignment (data not shown) and the occurrence of DAG kinase activities in prokaryotes (50) suggests that the yeast and bacterial DAG kinase homologues possess similar molecular, but perhaps not cellular, roles to those of their animal and plant homologues.

Significance of Domain Detection and Functional Prediction. Annotation of molecular function in sequence databases and even in the literature is difficult to interpret given that the term function may describe phenomena occurring at distinct levels, such as those of amino acids, domains, proteins, molecular complexes, cells, or organisms. Nevertheless, the examples shown above demonstrate that annotation of a certain domain can provide useful hints toward experimental characterization of function at different levels. Domain identification also might provide a counter-argument to a previously proposed molecular function. For example, identification of a PH domain and the absence of a detectable transmembrane region in a supposed integrin from C. albicans (Fig. 2g) argues strongly against its proposed role in cell adhesion (51). Integrins are transmembrane proteins that link the extracellular matrix with the cytoskeleton and normally contain, except for the B-4 subunit, short cytoplasmic sequences. The finding of a PH domain and high sequence similarity to S. cerevisiae BUD4 argues for its signaling role in bud site selection.

	DISCUSSION

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Many proteins are multidomain in character and possess multiple functions that often are performed by one or more component domains. A Web-based tool (SMART) has been designed that makes use of mainly public domain information to allow easy and rapid annotation of signaling multidomain proteins. The tool contains several unique aspects, including automatic seed alignment generation, automatic detection of repeated motifs or domains, and a protocol for combining domain predictions from homologous subfamilies. The ability of SMART to annotate single sequences or large datasets is exemplified by the cases described in Results, including annotation of the complete set of yeast ORFs.

Currently, large-scale or genome analysis is commonly performed by annotating ORFs with a single "best hit" from similarity searches. Ambiguities whether hits represent orthologs (i.e., homologues in different organisms that arose from speciation rather than intragenome duplication and are likely to have a corresponding function; ref. 52) or else paralogs (other members of multigene families) are not solved and omission of domain annotation also leads to misprediction of function. As most signaling proteins are multidomain in character, only annotation at the domain level avoids ambiguities in assigning homologies and functions to sequences, which may propagate further on additional findings of homology. Furthermore, deduction of the modular architecture is essential for the understanding of the complexities of multidomain eukaryotic signaling molecules; current annotation, however, does not adequately provide this information (Table 1). As examples of this, the existence of noncatalytic signaling domains cannot be deduced from the current yeast genome directory (8) and no human RasGEF domains currently are annotated in SwissProt. Graphical representation of the complement of modular proteins in a completed genome (e.g., the 622 signaling domains in 420 yeast proteins: http://www.bork.embl-heidelberg.de/Modules/syeast.html) might provide the basis for relating experimentally derived information concerning domains and multidomain proteins, to cellular events such as signaling.

Although other collections, such as PROSITE, Pfam, BLOCKS, and PRINTS, contain many more distinct domains or motifs, the focus of SMART on signaling allows significantly enhanced detection sensitivity, the inclusion of many families that are not represented in other collections, and offers a high level of specificity (i.e., a low rate of false positives that is essential for large-scale analysis). The SMART database shall be continually updated; alignment updates shall be semiautomated to avoid misalignments. Thus, forthcoming SMART database versions shall be hand-checked to provide datasets of high quality. In future, experimental findings that advance the understanding of domain structure and function also shall be provided via updates. As SMART is designed to obtain biologically relevant results without dependency on a single database search technique, there is potential to modify underlying methods to improve performance.