We submitted the sequence of S. cerevisiae
Rit1p (NCBI gene identification number 6323939) to the GeneSilico metaserver19
to predict the protein structure by fold-recognition methods. This analysis revealed that Rit1p is composed of two domains (roughly residues 1–340 and 341–512). The N-terminal domain (NTD) exhibited no evident similarity to any known protein structure or family. However, the C-terminal domain (CTD) of Rit1p was found to exhibit significant similarity to structures of dual-specificity phosphatases (DUSPs). Nearly all individual fold-recognition methods reported DUSP structures as the most preferred matches (e.g., HHSEARCH: 2 hcm_A, score 97.88, and 1wrm_A, score 97.28, with 100 being ideal match, and mGen-Threader: 1wrm_A, score 0.001, with 0 being ideal match). Based on these primary predictions, the consensus predictor PCONS unequivocally identified the DUSP fold as the only reasonable template for modeling: the top 10 matches exhibited scores in the range of 1.94–2.65, which indicate a highly confident prediction (see Methods for explanation of scores). The similarity between Rit1p and DUSPs was originally described in a B.Sc. thesis20
of the first author of this article, and it has been independently identified using HHsearch21
by Arcady Mushegian.22
DUSPs are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine or phosphothreonine residues, also within the same substrate. They have been implicated as major modulators of signaling pathways critical for cell growth and differentiation (reviewed in refs. 23–25
). They are often composed of multiple domains, with the conserved catalytic domains fused to other domains with various folds and functions.23
There are also known cases of DUSP-related proteins, such as MKSTYX, that lack the conserved residues in the active site and appear enzymatically inactive. While the evolutionary relationship and the resulting structural similarity between the CTD of Rit1p and DUSPs appears evident beyond any reasonable doubt, the question arises whether Rit1p possesses the DUSP-like active site and may exhibit a similar activity.
Based on the alignments from the metaserver, we have built a comparative model of the CTD () using the “FRankenstein's monster” modeling approach.26
Details of the modeling procedure and an explanation of the model assessment scores are presented in the Materials and Methods section. The best-scoring structure was evaluated as potentially “extremely good” by the PROQ method (predicted LGscore: 4.317), and the MetaMQAP method27
predicted that the model's root mean square deviation to the (currently unknown) true structure is around 3.4 Å. These values indicate that our structural prediction is likely to be correct and support the prediction that Rit1 has a common evolutionary ancestor with phosphatases. The model can, therefore, be used to make functional inferences at the level of individual residues (however, not with atomic precision) in connection with the analysis of sequence conservation in the Rit1p family.
A search of the non-redundant database at the NCBI revealed that Rit1 homologs are present in green plants, fungi and Giardia lamblia
, and that all members of this family possess both domains. Analysis of the multiple sequence alignment () reveals that the NTD is more conserved and contains several sequence motifs rich in invariant or nearly invariant residues, while the CTD contains two conserved motifs. One of the conserved motifs (hhhx-CxxGxDhS, where h indicates a hydrophobic residue and x a non-conserved residue) in the CTD corresponds to the active site motif of dual-specificity phosphatases. Interestingly, most (but not all) Rit1 family members possess a conserved Cys residue (C445 in S. cerevisiae
Rit1p) that corresponds to the catalytic Cys residue of DUSPs, which is used to form a cysteinyl-phosphate intermediate in the phosphate group transfer reaction.28
A catalytic Asp residue that fulfills a role of general acid in the reaction performed by DUSPs has no homologous counterpart in the Rit1 family. However, a spatially equivalent position in the putative phosphatebinding pocket is occupied by a conserved Asp residue (D450 in S. cerevisiae
Rit1p). This spatial equivalence, which became apparent only upon inspection of the model and was not obvious from the sequence alignment, suggests that the CTD of Rit1 proteins possesses a putative active site similar to those of DUSPs.
Figure 2 Multiple alignment of representative sequences from the Rit1 family and the known dual specificity phosphatase structure (2 hcm) identified by the fold-recognition analysis as the best template for modeling of the CTD. Secondary structures predicted for (more ...)