Residues included in the analysis at the different resolution levels.

<p>We focused on seven resolution levels of residues/codons within <i>S. cerevisiae</i> genes: 1) Structurally-determined interacting residues - the residues/codons involved in protein interactions, as reported in the 3DID database based on <i>S. cerevisiae</i> complexes solved by crystallography <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034503#pone.0034503-Stein2" target="_blank">[9]</a>. 2) Structurally-determined interacting domains - All residues/codons in yeast domains that were shown to mediate interaction in PPI structures solved by crystallography. 3) Residues in yeast domains inferred as mediating interactions - All residues/codons in domain-pairs in yeast PPIs, capable of mediating interaction. These domain-pairs were shown by crystallography to mediate PPIs in solved complex structures (not necessarily in yeast) and were projected onto yeast PPIs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034503#s3" target="_blank">Methods</a>). 4) Inferred interaction-mediating domains in yeast - All residues/codons in domains (not pairs) that were found in solved structures as capable of mediating PPIs in yeast. These include domains in proteins involved in PPIs and domains in proteins that were not yet shown to be involved in PPIs. 5) Inferred interaction-mediating domains - All residues/codons in domains (not domain-pairs) that were found in solved structures as involved in PPIs, not necessarily in yeast. 6) Domains - All residues/codons in domains (as opposed to extra-domain residues. 7) Proteins - Whole-protein residues (domains and extra-domains). (<b>A</b>) A schematic diagram describing the resolution levels. The figure is not scaled. (<b>B</b>) Schematic representation of the residues included in each resolution levels. The up panel describes the source of domains, interacting domains and interacting residues. The bottom panel shows schematically the residues/domains included in each resolution level and indicates the data source this classification is based on by the star color: red - data based on interacting domains in yeast proteins; blue - data based on interacting domains in other organisms; purple - data based on Pfam domains; pink - extra-domain regions.</p

Analysis of residue conservation in interacting domains.

<p>(<b>A</b>) Average fractions of non-synonymous mutations in residues determined by the various resolution levels (see caption of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034503#pone-0034503-g001" target="_blank">Figure 1</a>). A non-synonymous mutation was determined if there was a substitution in at least one strain, compared to the laboratory strain. (<b>B</b>) Comparison of pN/pS values between residues in a set determined by a resolution level and a complementary set of residues: 1) Interacting residues in yeast proteins based on solved structures were compared to non-interacting residues. 2) Yeast interacting domains based on solved structures were compared to non-interacting domains. 3) Domain-pairs in PPIs that can be mapped to structurally solved domain-domain interactions were compared to the other domains. 4) Domains documented in yeast as interacting were compared to domains that were not documented as interacting in solved structures in yeast. 5) Domains that were documented in yeast and other organisms as interacting domains were compared to domains that were not documented as interacting in solved structures in any organism. 6) Residues in protein domains were compared to residues that do not reside within domains (extra-domain regions). These six groups correspond to groups 1–6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034503#pone-0034503-g001" target="_blank">Figure 1</a>. g: residues in studied set. c: residues in complementary set. P-values of Kolmogorov-Smirnov test (applying FDR correction) are given at the bottom of each panel.</p

Coupling between phosphorylation events and domain-binding motifs.

<p>For each domain family (SH2, WW, PDZ and SH3), the bars denote the percent of motifs found to be phosphorylated either within or near them. Solid-colored and empty rectangular bars represent intra-motif phosphorylation and near-motif phosphorylation, respectively. All motifs are derived from the high reliability dataset, while phosphorylation events are derived from three data sets: LTP (low throughput evidence only), HTP (phosphorylation events based on evidence from high-throughput resources), and LTP+HTP (any type of evidence). Asterisks represent statistically-significant results (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#s4" target="_blank">Methods</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi-1002341-t001" target="_blank">Table 1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341.s003" target="_blank">Table S1</a>).</p

Mutually exclusive binding of domain pairs to the same protein segment.

<p>Summary of literature-documented double switches. The second column includes protein sequences, where residues vital for SH2 binding and residues vital for SH3/class I WW binding are in bold and underlined, respectively. Rows (1–3) describe experimentally-verified double switches. Rows (4–5) include examples for which there is evidence for the motif binding to each domain, but not for a direct switch. Note that Y534 in growth hormone receptor is phosphorylated according to a high-throughput experiment. Also note that evidence for Fyn-Cbl interaction exists for the Cbl (552–614) fragment (spanning 62 residues), where Y552 is the only tyrosine, suggesting that this tyrosine is bound by the SH2 domain in Fyn.</p

Phylogenetic traces of PDZ interaction-regulation unit evolution.

<p>This matrix summarizes the results for units of PDZ binding motifs and near-motif phosphorylation. The eukaryotic evolutionary tree is depicted above and left to the matrix (abbreviations below). The rows indicate the organism in which the motif probably appeared. The columns indicate the organism in which a potentially phosphorylated residue appeared. The order in which the motif and potentially phosphorylated residue appeared can thus be deduced from the matrix cells. For instance, the brown-framed cell represents the three cases in which the motif appeared in <i>D. melanogaster</i> and the potentially phosphorylated residue appeared in chicken. Accordingly, all cells below the diagonal (cyan) represent cases in which the potentially phosphorylated residue appeared after the motif. The diagonal cells represent cases in which the motif and the potentially phosphorylated residue appeared together. The cells above the diagonal represent cases in which the motif appeared after the potentially phosphorylated residue (red). Organism abbreviations: CHIMP- <i>p. troglodytes</i>, MOUSE- <i>m. musculus</i>, RATUS- <i>r. norvegicus</i>, BOVIN- <i>b. taurus</i>, CHICK- <i>g. gallus</i>, XENTR- <i>x. tropicalis</i>, DANRE- <i>d. rerio</i>, CIONA- <i>c. intestinalis</i>, DROME- <i>d. melanogaster</i>, ANOGA- <i>a. gambiae</i>, CAEEL- <i>c. elegans</i>, YEAST- <i>s. cerevisiae</i>, DICDI- <i>d. discoideum</i>, ARATH- <i>a. thaliana</i> and PLAFA- <i>p. falciparum</i>.</p

Frequency of various phylogenetic traces of motif-phosphorylation coupling.

<p>The stacked-bar graph details the relative frequency of the three possible phylogenetic traces of the interaction-regulation units (for either intra-motif phosphorylation or near-motif phosphorylation sites): (i) co-appearance of the motif and the potentially phosphorylated residue in the same organism (grey), (ii) the motif appeared before the potentially phosphorylated residue (cyan) (iii) the potentially phosphorylated residue appeared before the motif (red). For each domain we tested if the distribution of the various scenarios deviates from random by a χ² test. Asterisks denote statistically significant results (based on <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341.s008" target="_blank">Table S6</a>).</p

Experimental evidence of phosphorylation-mediated modulation of domain-motif interactions.

<p>*Numbers in parentheses indicate the distance between the motif and the proximal phosphorylation site/s.</p

Phosphorylation events as double switches.

<p>(<b>A</b>) A protein (black horizontal line) includes a segment that matches two sequence patterns: the first is typical for SH3 domain binding (green), and the second typifies SH2 domain binding (red). The non-phosphorylated form binds SH3 and not SH2 (upper), while phosphorylation inverts the binding preferences (lower). (<b>B</b>) Specificity switches within the PDZ domain family. A protein (black horizontal line) includes a segment that may bind distinct PDZ domains (upper). The non-phosphorylated form binds PDZ^a and not PDZ^b, while phosphorylation inverts these binding preferences (lower).</p

Dual sequence patterns used for the identification of potential double switches in human proteins.

<p>Column titles include sequence patterns for motifs that bind SH3 or class I WW domains (in red), and row titles include sequence patterns for motifs that bind different types of SH2 domains, upon motif phosphorylation (in blue). Each table cell includes a merged sequence pattern that hints at a dual binding potential of the motif to both SH2 and SH3 (or WW) domains. The columns under class I WW and SH3-1 titles represent the strict analysis scheme. Sequence patterns were extracted from the ELM database <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Gould1" target="_blank">[14]</a>. (i) An example for a dual motif. The PP.Y.N. sequence pattern is composed of the SH2^Grb2 Y.N. and the class I WW PP.Y patterns. (ii) Note that this sequence pattern encompasses seven positions.</p

Step-wise appearance of motifs and potential phosphorylation sites.

<p>(<b>A</b>) The motif is older than the potential phosphorylation site. The human CDK inhibitor 1B (top line) includes an SH3-binding motif (RxxK, highlighted in red) and a proximal tyrosine that may affect the motif's interaction potential upon phosphorylation <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Chu1" target="_blank">[74]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Harkiolaki1" target="_blank">[75]</a> (highlighted in cyan). The sequence pattern is conserved from <i>C. elegans</i> to human, but the tyrosine is conserved only between rat and human. This suggests that an old domain-binding motif has gained phospho-regulation in more recent organisms. Protein accessions are according to the Uniprot or Ensembl databases. (<b>B</b>) Potential phosphorylation site is older than the motif. The human Tau protein includes an SH3-binding motif (PxxP) and a proximal threonine that inhibits the motif's interaction potential upon phosphorylation <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Reynolds1" target="_blank">[76]</a>. This phosphorylation was also shown to induce a conformational change that unlocks the closed form of the protein <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Lin1" target="_blank">[77]</a>. The motif is conserved from <i>X. tropicalis</i> to human, while the potential phosphorylation site may have appeared earlier in evolution (present in <i>D. melanogaster</i>). This suggests that the domain-binding potential was established close to already functional phosphorylation.</p