Limits...
Evolution of the SH3 Domain Specificity Landscape in Yeasts.

Verschueren E, Spiess M, Gkourtsa A, Avula T, Landgraf C, Mancilla VT, Huber A, Volkmer R, Winsor B, Serrano L, Hochstenbach F, Distel B - PLoS ONE (2015)

Bottom Line: To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution.Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family.As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation-CRG, Barcelona, Spain.

ABSTRACT
To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a C. albicans Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for ~75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.

No MeSH data available.


Related in: MedlinePlus

Myo5 and Rvs167 binding validation.(A) Sepharose-bead bound GST or GST-tagged C-terminal myosin type I tails of ScMyo5 (984–1219), ScMyo3 (1010–1271), AgMyo5 (1084–1292), CaMyo5 (1004–1316) and SpMyo1 (1967–1217) were incubated with a total protein extract of S. cerevisiae supplemented with TRITC-labeled actin. The fluorescent halos around the beads (sized 50–150 μm) show the ability of the myosin type I tails of the four different yeast species to recruit active actin polymerization machinery to the beads while the negative control GST does not. Addition of 10 μM Latrunculin A inhibits actin polymerization. (B) Yeast two-hybrid strains co-transformed with the indicated bait and prey constructs were spotted (~104 cells) on minimal plates with histidine (His+), without histidine (His−), without histidine containing 2.5, 5, or 10 mM 3-amino-1,2,4-triazole (3AT), or without adenine (Ade–). Weak interactors activate only the HIS3 reporter and show growth on His−plates, while strong interactors activate both HIS3 and ADE2 reporters and show growth on His−plates containing 3AT or on Ade−plates. Note that CaRvs167-3 SH3 shows weak self-activation as revealed by growth on His−plates in the presence of an empty bait plasmid.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4466140&req=5

pone.0129229.g005: Myo5 and Rvs167 binding validation.(A) Sepharose-bead bound GST or GST-tagged C-terminal myosin type I tails of ScMyo5 (984–1219), ScMyo3 (1010–1271), AgMyo5 (1084–1292), CaMyo5 (1004–1316) and SpMyo1 (1967–1217) were incubated with a total protein extract of S. cerevisiae supplemented with TRITC-labeled actin. The fluorescent halos around the beads (sized 50–150 μm) show the ability of the myosin type I tails of the four different yeast species to recruit active actin polymerization machinery to the beads while the negative control GST does not. Addition of 10 μM Latrunculin A inhibits actin polymerization. (B) Yeast two-hybrid strains co-transformed with the indicated bait and prey constructs were spotted (~104 cells) on minimal plates with histidine (His+), without histidine (His−), without histidine containing 2.5, 5, or 10 mM 3-amino-1,2,4-triazole (3AT), or without adenine (Ade–). Weak interactors activate only the HIS3 reporter and show growth on His−plates, while strong interactors activate both HIS3 and ADE2 reporters and show growth on His−plates containing 3AT or on Ade−plates. Note that CaRvs167-3 SH3 shows weak self-activation as revealed by growth on His−plates in the presence of an empty bait plasmid.

Mentions: To experimentally confirm the conservation of the binding specificity of the type I myosin we chose an ex vivo approach established by Geli and colleagues [32]. This method assesses the ability of sepharose-bound proteins to induce actin polymerization using fluorescently labeled actin. We demonstrate that the SH3-containing C-terminal Myo5 tails of all four species were able to induce actin polymerization when incubated with total S. cerevisiae protein extract as revealed by a fluorescence halo formation around the sepharose beads (Fig 5A). As the interaction of the Myo5 SH3 domain with the Wiskott-Aldrich syndrome protein [WASP]-interacting protein (WIP) homolog Vrp1 was shown to be essential for the initiation of actin polymerization in S. cerevisiae [32], these data validate an interaction between ScVrp1 and all four Myo5 SH3 domains, confirming an interspecies conservation of the binding specificity of the type I myosins.


Evolution of the SH3 Domain Specificity Landscape in Yeasts.

Verschueren E, Spiess M, Gkourtsa A, Avula T, Landgraf C, Mancilla VT, Huber A, Volkmer R, Winsor B, Serrano L, Hochstenbach F, Distel B - PLoS ONE (2015)

Myo5 and Rvs167 binding validation.(A) Sepharose-bead bound GST or GST-tagged C-terminal myosin type I tails of ScMyo5 (984–1219), ScMyo3 (1010–1271), AgMyo5 (1084–1292), CaMyo5 (1004–1316) and SpMyo1 (1967–1217) were incubated with a total protein extract of S. cerevisiae supplemented with TRITC-labeled actin. The fluorescent halos around the beads (sized 50–150 μm) show the ability of the myosin type I tails of the four different yeast species to recruit active actin polymerization machinery to the beads while the negative control GST does not. Addition of 10 μM Latrunculin A inhibits actin polymerization. (B) Yeast two-hybrid strains co-transformed with the indicated bait and prey constructs were spotted (~104 cells) on minimal plates with histidine (His+), without histidine (His−), without histidine containing 2.5, 5, or 10 mM 3-amino-1,2,4-triazole (3AT), or without adenine (Ade–). Weak interactors activate only the HIS3 reporter and show growth on His−plates, while strong interactors activate both HIS3 and ADE2 reporters and show growth on His−plates containing 3AT or on Ade−plates. Note that CaRvs167-3 SH3 shows weak self-activation as revealed by growth on His−plates in the presence of an empty bait plasmid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466140&req=5

pone.0129229.g005: Myo5 and Rvs167 binding validation.(A) Sepharose-bead bound GST or GST-tagged C-terminal myosin type I tails of ScMyo5 (984–1219), ScMyo3 (1010–1271), AgMyo5 (1084–1292), CaMyo5 (1004–1316) and SpMyo1 (1967–1217) were incubated with a total protein extract of S. cerevisiae supplemented with TRITC-labeled actin. The fluorescent halos around the beads (sized 50–150 μm) show the ability of the myosin type I tails of the four different yeast species to recruit active actin polymerization machinery to the beads while the negative control GST does not. Addition of 10 μM Latrunculin A inhibits actin polymerization. (B) Yeast two-hybrid strains co-transformed with the indicated bait and prey constructs were spotted (~104 cells) on minimal plates with histidine (His+), without histidine (His−), without histidine containing 2.5, 5, or 10 mM 3-amino-1,2,4-triazole (3AT), or without adenine (Ade–). Weak interactors activate only the HIS3 reporter and show growth on His−plates, while strong interactors activate both HIS3 and ADE2 reporters and show growth on His−plates containing 3AT or on Ade−plates. Note that CaRvs167-3 SH3 shows weak self-activation as revealed by growth on His−plates in the presence of an empty bait plasmid.
Mentions: To experimentally confirm the conservation of the binding specificity of the type I myosin we chose an ex vivo approach established by Geli and colleagues [32]. This method assesses the ability of sepharose-bound proteins to induce actin polymerization using fluorescently labeled actin. We demonstrate that the SH3-containing C-terminal Myo5 tails of all four species were able to induce actin polymerization when incubated with total S. cerevisiae protein extract as revealed by a fluorescence halo formation around the sepharose beads (Fig 5A). As the interaction of the Myo5 SH3 domain with the Wiskott-Aldrich syndrome protein [WASP]-interacting protein (WIP) homolog Vrp1 was shown to be essential for the initiation of actin polymerization in S. cerevisiae [32], these data validate an interaction between ScVrp1 and all four Myo5 SH3 domains, confirming an interspecies conservation of the binding specificity of the type I myosins.

Bottom Line: To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution.Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family.As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation-CRG, Barcelona, Spain.

ABSTRACT
To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a C. albicans Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for ~75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.

No MeSH data available.


Related in: MedlinePlus