Combining multiple structure and sequence alignments to improve sequence detection and alignment: application to the SH2 domains of Janus kinases

Abstract

In this paper, an approach is described that combines multiple structure alignments and multiple sequence alignments to generate sequence profiles for protein families. First, multiple sequence alignments are generated from sequences that are closely related to each sequence of known three-dimensional structure. These alignments then are merged through a multiple structure alignment of family members of known structure. The merged alignment is used to generate a Hidden Markov Model for the family in question. The Hidden Markov Model can be used to search for new family members or to improve alignments for distantly related family members that already have been identified. Application of a profile generated for SH2 domains indicates that the Janus family of nonreceptor protein tyrosine kinases contains SH2 domains. This conclusion is strongly supported by the results of secondary structure-prediction programs, threading calculations, and the analysis of comparative models generated for these domains. One of the Janus kinases, human TYK2, has an SH2 domain that contains a histidine instead of the conserved arginine at the key phosphotyrosine-binding position, betaB5. Calculations of the pK(a) values of the betaB5 arginines in a number of SH2 domains and of the betaB5 histidine in a homology model of TYK2 suggest that this histidine is likely to be neutral around pH 7, thus indicating that it may have lost the ability to bind phosphotyrosine. If this indeed is the case, TYK2 may contain a domain with an SH2 fold that has a modified binding specificity.

Figure 1

Alignment of JAKs and 19 SH2 domains of known structures. The multiple alignment of the SH2 domains is structure-based (6), whereas the four JAKs are aligned with an HMM of the SH2 domain family (see text for details). Minor manual modifications were made to close gaps in predicted secondary structures, e.g., the αB helix of TYK2. The conventional secondary-structure assignments and numbering for SH2 domains (12) are illustrated in cartoon form above the alignment. Residues are colored according to secondary structure: gold, β-strand; magenta, α-helix. For the JAKs, the secondary structure displayed is that predicted by jpred2 (39). For the 19 SH2 domains, the DSSP (49) secondary-structure assignments from the PDB coordinates are shown.

Figure 2

Flowchart of the procedure used to align all SH2 domains.

Figure 3

Sequence- and structure-based alignments of SH2 domains. (a) Alignments of several SH2 domains of known structure (colors indicate secondary structure elements, as in Fig. 1). (b) Alignments of Cbl with Src-SH2. Residues within Cbl-SH2 are shown in bold.

Figure 4

3D profiles and cumulative scores for JAK-SH2 models and 19 SH2 structures. Profiles were obtained from the Verify3D server (37). (a) 3D profiles of the 19 selected SH2 domain structures and of the Tyk2-SH2 model. (b) Cumulative 3D scores for the 19 selected SH2 domain structures and of the four JAK models.