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.
Highlights
Peptide recognition modules, like the Src homology 3 (SH3) domains, bind peptide motifs with low affinity and are predominantly found in signaling pathways, where they mediate transient protein-protein interactions that regulate cell proliferation and differentiation
We found that all S. cerevisiae SH3 proteins could be mapped to orthologs in, A. gossypii, C. albicans, and S. pombe (S1 Table)
To facilitate the cross-species comparison in this study, from here on we will refer to homologs in A. gossypii (Ag), C. albicans (Ca), and S. pombe (Sp) with names according to their S. cerevisiae (Sc) based family names
Summary
Like the Src homology 3 (SH3) domains, bind peptide motifs with low affinity and are predominantly found in signaling pathways, where they mediate transient protein-protein interactions that regulate cell proliferation and differentiation. These recognition domains often bind a core motif common to the domain family, surrounded by a number of specificity-determining residues that minimize cross-reactivity. SH3 domains generally bind to proline-rich sequences containing a core PXXP motif (where X is any amino acid) flanked by a positively charged residue [1,2]. Recent studies, which capitalize on the continuous advances in high-throughput phage-display library development and sequencing technologies, generated up to 10 billion random peptides to explore the SH3 domain recognition landscape in an unbiased fashion and confirmed its complexity [9,10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
