To illustrate some of the possible sequence searches, alignments, and analyses that can be carried out via the Web, we will use two examples: the catalytic domain from a PI3-kinase and the protein-interaction domain SH2. Structural information and an accurate alignment in the BAliBase database are available for the family of SH2 domains.
The human Syk tyrosine kinase carboxy-terminal SH2 domain is the first query sequence. Protein searches with BLAST through the SWISS-PROT database gave 149 sequences below the default £-value cut-off. All these were SH2-related domains. That is a lot of information to cope with. All the E-values were very low, indicating that all the hits were significant. This is a case of result overload. Decreasing the E- value cut-off would have no effect in this case, as all the hits were far below the threshold used.
Figure 4.12
The results of a search of the SWISS-PROT protein sequence database using BLAST with PI3-kinase plOOoc as the query sequence. (A) Output from a standard BLAST search. Each line reports a separate database sequence. The penultimate column gives the alignment score, and the last column the E- value. Hits before the arrow are significant, while below the arrow the hit does not have a
significant score. (B) A BLAST search on one month's new sequences, using the same query sequence as in (A), finds only two matches. One is a PI4-kinase, which has most of its sequence aligned to the query sequence (magenta line). The other has only a small region aligned (black line) and a borderline score. (C) Output from a Conserved Domain Database (CDD) search.
To reduce the large number of hits one could search a subset of the data, for example only the newest sequences in the database (the "month" option; that is, those deposited in the last month) or a specific genome database. A search through sequences released in the past month detected eight sequences, all with significant scores, of which three had not been identified. A search through the Drosophila genome data yielded three hits, all of which are unknown. Taking one of the regions that matched our SH2 (a section of the Drosophila 3R chromosome arm) and searching with this sequence through SWISS-PROT yielded a highly significant hit to an SH2 domain of a rat protein. So we may have identified a previously unknown Drosophila sequence as containing an SH2 domain.
This example illustrates a search through the database with a family that is very well represented and shows the problems that can arise. We will now look at a family that is not so well represented—the PI3-kinases.
First the SWISS-PROT database was searched using the catalytic domain protein sequence from the PI3-kinase pi 10a using BLAST with the £-value cut-off set to 1. Thirty-two hits were found. In this list there are three near-identical isoforms of pi 10a which have an £-value of 0.0; that is, the chance of obtaining such a match with random sequence is taken to be zero. There is one match that is not significant: ribonu- cleoside-diphosphate reductase, with an E-value of 0.59 (see Figure 4.12A). From the assigned function this is clearly a different enzyme, but the enzymatic reactions of both this reductase and the kinases involve a nucleotide, which might have led to some small degree of similarity between the sequences. Any such speculation would need further and more thorough investigation. If we rerun the search with the E- value cut- off set to 0.01 (the advised setting) only the significant matches are retrieved.
Searching with BLAST through a subset of sequences such as those that have only been released in one month found two hits: one is a homolog of PI3-kinase, a PI4- kinase with a significant £-value, and the other is a segment of an Arabidopsis thaliana protein, atRad3, with an E-value score of borderline significance. From the length of the matched sequence illustrated in the search output (see Figure 4.12B) the segment seems far too short to be of interest; compare the length of the matched PI4- kinase, in magenta. For this reason the hit can now be reclassified as not significant. Another useful option available within the BLAST search server is a concurrent search of the Conserved Domain Database (CDD) entries. Figure 4.12C shows the results of using this option on the PI3-kinase sequence. Proteins often contain several domains, and the program CD-Search can potentially identify domains present in a protein sequence. CDD contains domains derived mainly from the SMART and Pfam protein-family databases. To identify conserved domains in a protein sequence, the CD method uses the BLAST algorithm where the query sequence is matched with a PSSM designed from the conserved domain align- ments. Matches are shown as either a pairwise alignment of the query sequence to a representative domain sequence or as a multiple alignment.
A FASTA search with the pi 10a sequence through SWISS-PROT with default settings (k-tuple = 2) yielded 36 hits, of which eight had a nonsignificant score (see Figure 4.13). Of these eight, ribonucleoside-diphosphate reductase was also found by BLAST. Although both FASTA and BLAST report an f-value, the actual values are different, which reflects subtle differences in the methods used. An SSEARCH search of the SWISS-PROT database with default settings found 29 significant hits. SSEARCH, a more rigorous method, found fewer hits than BLAST or FASTA.