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Mapping of interaction sites of the Schizosaccharomyces pombe protein Translin with nucleic acids and proteins: a combined molecular genetics and bioinformatics study.

Eliahoo E, Ben Yosef R, Pérez-Cano L, Fernández-Recio J, Glaser F, Manor H - Nucleic Acids Res. (2010)

Bottom Line: TRAX is a Translin paralog associated with Translin, which has coevolved with it.Similar nucleic acid and protein interaction sites were also predicted for the human Translin.Thus, our results appear to generally apply to the Translin family of proteins, and are expected to contribute to a further elucidation of their functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.

ABSTRACT
Translin is a single-stranded RNA- and DNA-binding protein, which has been highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. TRAX is a Translin paralog associated with Translin, which has coevolved with it. We generated structural models of the S. pombe Translin (spTranslin), based on the solved 3D structure of the human ortholog. Using several bioinformatics computation tools, we identified in the equatorial part of the protein a putative nucleic acids interaction surface, which includes many polar and positively charged residues, mostly arginines, surrounding a shallow cavity. Experimental verification of the bioinformatics predictions was obtained by assays of nucleic acids binding to amino acid substitution variants made in this region. Bioinformatics combined with yeast two-hybrid assays and proteomic analyses of deletion variants, also identified at the top of the spTranslin structure a region required for interaction with spTRAX, and for spTranslin dimerization. In addition, bioinformatics predicted the presence of a second protein-protein interaction site at the bottom of the spTranslin structure. Similar nucleic acid and protein interaction sites were also predicted for the human Translin. Thus, our results appear to generally apply to the Translin family of proteins, and are expected to contribute to a further elucidation of their functions.

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Space filling (A) and ribbon representation (B) of the spTranslin front face displaying the substitution and truncation mutations generated and analyzed in the present study. Substitution of residues stained in red caused the strongest reduction in the affinities towards the RNA or DNA probe (see Table 1 for details). Substitution of residues stained in yellow and green caused partial and no inhibition, respectively, in binding to the probes. Substitution of residues colored violet caused an increase in the affinity for single-stranded DNA. The light blue amino acid residues at the carboxy terminus were missing from the truncation variant designated Y228stop (see Table 2 and the text).
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Figure 4: Space filling (A) and ribbon representation (B) of the spTranslin front face displaying the substitution and truncation mutations generated and analyzed in the present study. Substitution of residues stained in red caused the strongest reduction in the affinities towards the RNA or DNA probe (see Table 1 for details). Substitution of residues stained in yellow and green caused partial and no inhibition, respectively, in binding to the probes. Substitution of residues colored violet caused an increase in the affinity for single-stranded DNA. The light blue amino acid residues at the carboxy terminus were missing from the truncation variant designated Y228stop (see Table 2 and the text).

Mentions: A more detailed inspection of the OPRA and PFP results reveals that the patches predicted by both algorithms are unusually rich in arginine residues (see also Figures 4 and 8). The OPRA highest scores, which peak at the left corner of the patch, encompass three arginine residues: R210 and R211 (score 4) and R222 (score 3). The PFP-predicted highest scores, which peak on the right side of the equatorial patch, include residues R35 and R102 (score 4) and R211 (score 3). The four highest scored arginines in both methods, R35, R102, R210 and R211 were, in fact, experimentally observed to play a significant role in nucleic acid binding (see the next section of the Results and Table 1). It should also be noted that there is an overlap of both predictions in the lower part of their respective patches. This overlap includes residuesY94 and K95 (in light blue and cyan), which have lower overall scores, but were also shown experimentally to be involved in nucleic acid binding. Finally, it is noteworthy that the two methods are subject to different types of errors. The PFP predictions are highly dependent upon the model structures, and are, therefore, subject to modeling errors. OPRA is much less dependent on the specific model geometries and is, therefore, less sensitive to modeling errors (see Supplementary Figures S3 and S4). However, the OPRA scores are based on limited statistics of existing non-redundant PDB complexes of protein–RNA, while proteins related to Translin may be underrepresented in the OPRA statistics. Thus, the strength of our predictions stems from the use of these two different bioinformatics methods and 14 models. Indeed, the predictions seem to agree well with experimental data presented in the section ‘Experimental verification of the bioinformatics predictions regarding the functional regions in spTranslin’.Figure 4.


Mapping of interaction sites of the Schizosaccharomyces pombe protein Translin with nucleic acids and proteins: a combined molecular genetics and bioinformatics study.

Eliahoo E, Ben Yosef R, Pérez-Cano L, Fernández-Recio J, Glaser F, Manor H - Nucleic Acids Res. (2010)

Space filling (A) and ribbon representation (B) of the spTranslin front face displaying the substitution and truncation mutations generated and analyzed in the present study. Substitution of residues stained in red caused the strongest reduction in the affinities towards the RNA or DNA probe (see Table 1 for details). Substitution of residues stained in yellow and green caused partial and no inhibition, respectively, in binding to the probes. Substitution of residues colored violet caused an increase in the affinity for single-stranded DNA. The light blue amino acid residues at the carboxy terminus were missing from the truncation variant designated Y228stop (see Table 2 and the text).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875027&req=5

Figure 4: Space filling (A) and ribbon representation (B) of the spTranslin front face displaying the substitution and truncation mutations generated and analyzed in the present study. Substitution of residues stained in red caused the strongest reduction in the affinities towards the RNA or DNA probe (see Table 1 for details). Substitution of residues stained in yellow and green caused partial and no inhibition, respectively, in binding to the probes. Substitution of residues colored violet caused an increase in the affinity for single-stranded DNA. The light blue amino acid residues at the carboxy terminus were missing from the truncation variant designated Y228stop (see Table 2 and the text).
Mentions: A more detailed inspection of the OPRA and PFP results reveals that the patches predicted by both algorithms are unusually rich in arginine residues (see also Figures 4 and 8). The OPRA highest scores, which peak at the left corner of the patch, encompass three arginine residues: R210 and R211 (score 4) and R222 (score 3). The PFP-predicted highest scores, which peak on the right side of the equatorial patch, include residues R35 and R102 (score 4) and R211 (score 3). The four highest scored arginines in both methods, R35, R102, R210 and R211 were, in fact, experimentally observed to play a significant role in nucleic acid binding (see the next section of the Results and Table 1). It should also be noted that there is an overlap of both predictions in the lower part of their respective patches. This overlap includes residuesY94 and K95 (in light blue and cyan), which have lower overall scores, but were also shown experimentally to be involved in nucleic acid binding. Finally, it is noteworthy that the two methods are subject to different types of errors. The PFP predictions are highly dependent upon the model structures, and are, therefore, subject to modeling errors. OPRA is much less dependent on the specific model geometries and is, therefore, less sensitive to modeling errors (see Supplementary Figures S3 and S4). However, the OPRA scores are based on limited statistics of existing non-redundant PDB complexes of protein–RNA, while proteins related to Translin may be underrepresented in the OPRA statistics. Thus, the strength of our predictions stems from the use of these two different bioinformatics methods and 14 models. Indeed, the predictions seem to agree well with experimental data presented in the section ‘Experimental verification of the bioinformatics predictions regarding the functional regions in spTranslin’.Figure 4.

Bottom Line: TRAX is a Translin paralog associated with Translin, which has coevolved with it.Similar nucleic acid and protein interaction sites were also predicted for the human Translin.Thus, our results appear to generally apply to the Translin family of proteins, and are expected to contribute to a further elucidation of their functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.

ABSTRACT
Translin is a single-stranded RNA- and DNA-binding protein, which has been highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. TRAX is a Translin paralog associated with Translin, which has coevolved with it. We generated structural models of the S. pombe Translin (spTranslin), based on the solved 3D structure of the human ortholog. Using several bioinformatics computation tools, we identified in the equatorial part of the protein a putative nucleic acids interaction surface, which includes many polar and positively charged residues, mostly arginines, surrounding a shallow cavity. Experimental verification of the bioinformatics predictions was obtained by assays of nucleic acids binding to amino acid substitution variants made in this region. Bioinformatics combined with yeast two-hybrid assays and proteomic analyses of deletion variants, also identified at the top of the spTranslin structure a region required for interaction with spTRAX, and for spTranslin dimerization. In addition, bioinformatics predicted the presence of a second protein-protein interaction site at the bottom of the spTranslin structure. Similar nucleic acid and protein interaction sites were also predicted for the human Translin. Thus, our results appear to generally apply to the Translin family of proteins, and are expected to contribute to a further elucidation of their functions.

Show MeSH