Conservation Study of the SH3 Domains of <i>S. cerevisiae</i> in Ten Other Yeast Genomes

<p>CD, conserved domain (the SH3-containing protein has an ortholog and the ortholog SH3 domain is possibly conserved, i.e., less than three conservative changes and no nonconservative changes in the binding positions); DD, divergent domain (SH3-containing protein has an ortholog in this genome but the domain is not on the same branch of the phylogenetic tree); NO, no ortholog (no ortholog found for SH3-containing protein in a particular genome); PD, possibly divergent (SH3-containing protein has an ortholog in this genome but the ortholog SH3 domain has at least one nonconservative change in the binding positions or more than two conservative changes in the binding positions).</p

Size of Probing Window When Looking for Conservation of the Consensus Sequence in Orthologs of the Putative Target Protein

<p>We defined the conservation score as simply the number of species where the consensus sequence is conserved. With this information the accuracy and coverage were calculated, with the gold (A) and platinum (B) positive sets, for consensus sequence conserved in different numbers of species and for different sizes of the probing region.</p

Optimal Divergence Time to Search for Conservation of Target Motif of SH3 Domains

<p>We designated seven groups of species with an increasing average divergence time from <i>S. cerevisiae</i> and calculated for each group the highest accuracy obtained for restricted windows of coverage. We used the gold positive and the negative set to calculate the accuracy and coverage (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#s3" target="_blank">Materials and Methods</a>). The seven groups of species are as follows: (1) <i>S. bayanus, S. paradoxus, S. mikatae,</i> and <i>C. glabrata</i> (average divergence of 112.5 My from <i>S. cerevisiae</i>); (2) <i>S. paradoxus, S. mikatae, C. glabrata,</i> and <i>K. lactis</i> (average divergence of 200 My from <i>S. cerevisiae</i>); (3) <i>S. mikatae, C. glabrata, K. lactis,</i> and <i>C. albicans</i> (average divergence of 387.5 My from <i>S. cerevisiae</i>); (4) <i>C. glabrata, K. lactis, C. albicans,</i> and <i>D. hansenii</i> (average divergence of 575 My from <i>S. cerevisiae</i>); (5) <i>K. lactis, C. albicans, D. hansenii,</i> and <i>Y. lipolytica</i> (average divergence of 725 My from <i>S. cerevisiae</i>); (6) <i>C. albicans, D. hansenii, Y. lipolytica,</i> and <i>N. crassa</i> (average divergence of 875 My from <i>S. cerevisiae</i>); and (7) <i>D. hansenii, Y. lipolytica, N. crassa,</i> and <i>Sch. pombe</i> (average divergence of 950 My from <i>S. cerevisiae</i>). The individual values for the divergence time from <i>S. cerevisiae</i> were taken from the literature [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#pcbi-0010026-b32" target="_blank">32</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#pcbi-0010026-b42" target="_blank">42</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#pcbi-0010026-b43" target="_blank">43</a>]. Although we tried to create groups that would not have genomes of species with very different separation dates from <i>S. cerevisiae,</i> it should be noted that because of the small number of available genomes, the groups are not homogenous. Also, the values of the divergence time of each species were not always obtained with the same method. Therefore, this range of values should be viewed critically.</p

Most Informative Genomes in the Search for Conservation of Target Motif of SH3 Domains

<p>We created all possible combinations of two or more genomes of our set of ten genomes. For each combination we calculated the highest accuracy obtained for 11 windows of coverage from 15% to 70% at intervals of 5%. We then calculated the average frequency, over all coverage windows, of each individual species in all groups of genomes, in the combinations of genomes scoring within the 20% highest accuracy values and in the combinations scoring in the lowest 20% values of accuracy. We then used a <i>t</i>-test to determine, for each species, whether the average frequencies within the highest and lowest combinations were significantly different from the frequency in all possible combinations. *, <i>p <</i> 0.05; **, <i>p <</i> 0.001.</p

Predictions of <i>S. cerevisiae</i> SH3 Interactions

<p>We considered that a potential target consensus sequence, found by pattern matching, in an <i>S. cerevisiae</i> protein would be biologically relevant if it was within an unstructured region of the <i>S. cerevisiae</i> protein and also conserved in four of the seven comparison genomes used. <i>(C. glabrata, K. lactis, C. albicans, D. hansenii, Y. lipolytica, N. crassa,</i> and <i>Sch. pombe)</i>. Red lines indicate the interactions for which we found some experimental evidence in protein interaction databases [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#pcbi-0010026-b59" target="_blank">59</a>–<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#pcbi-0010026-b61" target="_blank">61</a>]; thin black lines indicate interactions between proteins that are labeled as locating to different compartments; thick black lines indicate interactions for which we found no evidence. There were two <i>S. cerevisiae</i> SH3 domains for which we could not predict any interaction because of the stringency applied. A complete list of the interactions with function, localization, and binding positions is given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0010026#pcbi-0010026-st004" target="_blank">Table S4</a>.</p

Protein Binding Domains with Many Structural Interactions Observed Have a Higher Link Turnover

<p>We have grouped proteins containing domains with increasing observed structural interactions with other domain types and calculated for each bin the rate of change of interactions. Proteins containing domains known to interact with many other different domains have a higher rate of change of interactions than proteins containing domains with few known interactions.</p

Combining Conservation and Secondary Structure Prediction

<p>We calculated, with the gold (A) and platinum (B) positive sets, the accuracy and coverage for target prediction when including or excluding secondary structure information. We used a probing region of 210 alignment positions in this analysis.</p

Preferential Interaction Turnover Is Observed in All Eukaryotic Interactomes

<p>We have binned proteins according to their average number of interactions and calculated for each bin the rate of change of interactions. There is a very strong correlation between the degree of connectivity and the interaction turnover.</p

Map of the Constructs Used in This Study

<p>The repressor binding sites overlap with T7 or SP6 promoters and vary between constructs. In this way, it is possible to alter the connectivity of the repressive interactions by the products of genes A, B, and C. Repressive interactions are denoted by T-bars. The start codon of each gene is in Kozak context and is denoted by “GCC ATG G.”</p

Varying Diffusion and Degradation Parameters

<div><p>Computer model of gene network, scaled to <i>Drosophila</i> length (0.5 mm). Diffusion parameters are varied for mRNA (Dm), protein (Dp), and T7 activator (DX). Data are plotted as percentage of total output protein (y-axes) against chamber position (x-axes), for 10-min simulations.</p> <p>(A) Outputs for protein A.</p> <p>(B) Output for protein B. Graphs with “target behaviour” are shaded grey, and the four asterisks mark the parameter sets used to generate outputs for proteins A and C.</p> <p>(C) Outputs for protein C.</p> <p>(D) Effect of adding protease degradation to B-output, shown at 15-min intervals, over a 2.5-h time course (parameters: DX = 0.43 μm²s⁻¹; Dm = Dp = 0.02 μm²s⁻¹; t_1/2 = 770 s).</p></div