Identification of yeast WW domain-protein interactions
We used protein microarrays to generate a protein interaction map of yeast WW domain-containing proteins. The microarrays were constructed by printing 4,088 proteins from S. cerevisiae in duplicate on nitrocellulose-coated glass slides. Other proteins printed on the arrays served as controls, including biotinylated antibodies for the detection of the biotinylated probes and gluthathione S-transferase for the analysis of binding specificity. In Y2H experiments with several of these WW domains present in DNA-binding domain fusions as either full-length proteins or isolated domains, we were unsuccessful in recovering previously reported interactions and unable to test many of the constructs due to their transcriptional self-activation (data not shown). Therefore, protein microarrays provided an alternative method to identify the protein interaction partners of these domains.
We expressed each of the individual domains in Escherichia coli
as a fusion to either glutathione S-transferase (GST) or maltose binding protein, and purified the fusion proteins (Figure ). During purification, WW domain fusion proteins were biotinylated using an amine-reactive biotinylation reagent, and each of the purified domains was used to probe duplicate protein microarrays. We were unable to obtain sufficient expression of either type of fusion protein containing the WW domain from Alg9, and thus focused on the remaining 12 WW domain probes. Protein-protein interactions on the microarrays were detected by the addition of fluorophore-conjugated streptavidin, and individual spots on the microarray were visualized by fluorescence scanning (Figure ). Previously, protein-protein and protein-lipid interactions identified using protein microarrays were shown to be highly reproducible [31
]. However, because of the importance of reproducibility in any protein interaction experiment, we applied each probe protein to two separate microarrays. After data processing, only those proteins found as high-confidence interactions were selected for further analysis. We defined high-confidence interactions to be those in which four independent observations of the interaction were made (that is, signals greater than three standard deviations above the mean spot fluorescence for a protein printed in duplicate on two separate microarrays). To identify interactions that might be platform-specific, we compared our initial data to a set of 13 supplementary protein microarray experiments that had previously been carried out (GAM, unpublished data). We removed 15 proteins from our data set that were found in more than half of these experiments, leaving 587 high-confidence interactions between 12 WW domains and 207 proteins (Additional data file 1).
Figure 2 Purification of WW domain fusion proteins. Coomassie-stained SDS-PAGE gel of WW domain fusion proteins following protein purification (top panels), western blot detection of fusion protein expression with anti-GST antibody (left middle panel) or anti-myc (more ...)
Figure 3 Protein microarray data and the Rsp5 network. (a) A microarray was probed with the first WW domain from Rsp5 and interactions were visualized via application of dye-labeled streptavidin and fluorescent scanning. Following data processing, two proteins (more ...)
Properties of the WW domain network
Within this network, the number of interactions observed with different WW domain probes varied from 86 interactions for the third WW domain of Rsp5 to 7 for Vid30 (Figure ); a recent study of a human 14-3-3 protein using protein microarrays identified 20 proteins as 14-3-3 interactors [32
]. The three domains from Rsp5 together interacted with 124 proteins (about 60% of the network), 45 of which were identified solely by these domains (Figure ). Conversely, the first domain from Prp40 interacted with one protein uniquely and the domain from Set2 had no unique partners. In general, there is a large degree of overlap within the network, as 53 proteins were found by at least 4 different domain probes.
Figure 4 WW domain network properties. (a) The number of interaction partners identified using each WW domain probe. (b) Log-log plot of the node degree distribution within the WW domain network. Black circles represent WW domain probes and red circles represent (more ...)
We used the Gene Ontology (GO) hierarchy [33
] to identify regions of the network that are enriched for particular classifications. The network was first split into 12 subnetworks, each consisting of a single WW domain probe and its interaction partners. These subnetworks contain a number of significant (P
< 0.05 using a hypergeometric test) enrichments of GO annotations (Additional data file 2). In particular, an enrichment of proteins involved in cofactor metabolism suggests a role for Rsp5 in the assembly or localization of the biosynthetic enzymes responsible for the metabolism of thiamine and other cofactors (Figure ). Enrichment of proteins within the network that localize to the peroxisome suggests that Rsp5, Ssm4 and Prp40 may be involved in processes within this organelle. Proteins containing WW domains also affect the localization and degradation of several proteins from the ER and other membranous intracellular compartments. For example, deletion of Ssm4 abrogates degradation of the ER transmembrane protein Ubc6 [18
], and Rsp5-mediated ubiquitination of plasma membrane proteins directs their internalization and targeting to the endosomal-lysosomal pathway [17
]. In addition, we observe interactions with several other ER proteins (for example, Rsp5 interacts with Ubc6 and Pdi1) and GTP-hydrolyzing proteins involved in vesicle transport (for example, Ssm4 interacts with Ypt6 and Ess1 interacts with Ypt53).
Protein-protein interaction networks have a common underlying topology in which the distribution of node degrees can be fit to a power law [34
]. Intuitively, this observation is consistent with protein functions: many proteins are specialized and interact with relatively few partners, whereas relatively few proteins are involved in numerous processes and interact with many partners. However, discrepancies can arise when this analysis is applied to small, sampled subsets of larger networks [35
]. Our interaction network differs from existing networks because it is focused on a single type of protein domain, and is likely, therefore, to be more heavily sampled (that is, more locally complete) than previous large-scale screens. The node degree distribution of the WW domain network exhibits the expected 'scale-free' topology of protein interaction networks (Figure ).
We searched the network for groups of proteins having conserved protein domains from the eMotif database [36
], but found no significantly enriched protein domains except for the WW domain itself (data not shown). This observation is consistent with the fact that binding sites recognized by WW domains are short primary sequences as opposed to sizable protein domains. We also used data compiled for Y2H and AP-MS experiments available from the Saccharomyces
Genome Database [29
] to identify 19 proteins within the network that have not been reported as having known protein interaction partners (Figure ). Analysis of these proteins using the GO Term Finder available from the Saccharomyces
Genome Database indicates no consistent functional annotation within this set of proteins.
Venn diagram illustrating the representation of yeast proteins involved in protein-protein interactions found using yeast two-hybrid (Y2H) assay, protein epitope-tag affinity purification/mass spectrometry (AP-MS) and protein microarray strategies.
Within the interaction network generated in this study, a total of 13 interactions have support from experimental studies, bioinformatic approaches, or both. Eight interactions have been observed previously by either the Y2H assay [3
] or AP-MS [5
]. Five of these involved the ubiquitin ligase Rsp5, which targets multiple proteins for degradation [37
], two involve interactions with Prp40, and the final one is the interaction between Ess1 and Bcy1, a regulatory subunit of cAMP-dependent protein kinase A [5
]. Two interactions involving Rsp5 were found in a recent screen for Rsp5 substrates [38
]. A probabilistic network of functional linkages [1
] supports eight interactions that we identified (Additional data file 3). We searched for orthologous interactions ('interologs' [39
]) between our dataset and the recently generated protein interaction maps of Drosophila melanogaster
], Caenorhabditis elegans
] and Homo sapiens
] but found no conserved interactions.
Given the low degree of overlap between these protein microarray data and existing datasets, validation of these interactions by other approaches is an important step prior to further analysis of the biology of these interactions. For example, a reversed microarray experiment could be used to address array-based artifacts, in which microarrays would be assembled using the WW domain-fusion proteins as array features, and these arrays would be probed with the interacting proteins that were originally identified. Alternatively, epitope-tagged versions of the WW domains could be introduced into cells, and interacting proteins would be identified using immunoprecipitation and western blotting or affinity purification and mass spectrometry; a similar strategy was used to identify proteins that interact with human WW domain-containing proteins [43
WW ligand sequence motif representation
To address ligand specificity, we compiled a list of primary sequence motifs of known WW domain-ligands from the literature and searched the proteins in our network for occurrences of these motifs. Within the network, 28 proteins have canonical PY motifs and 5 have poly-proline motifs. Twenty-six proteins have PPR motifs, and 38 proteins have a degenerate PY motif, the LPxY motif, which was previously shown to be a determinant for Rsp5 specificity [44
]; 24 of these 38 interacted with Rsp5 (Figure ). Twenty proteins have more than one motif or possess motifs from multiple classes (Additional data file 4). We found a significant enrichment of proteins with PY and LPxY motifs (P
and 0.02, respectively, using a binomial test) relative to all proteins present on the microarrays. In the S. cerevisiae
proteome, approximately 250 proteins contain PY motifs (4% of all proteins) and 400 proteins contain LPxY motifs (7%). In contrast, approximately 30% of the proteins in the WW domain network contain either PY or LPxY motifs.
The prevalence of the PY motif within the network is expected given the group I classification of the three WW domains from Rsp5. Of the 124 proteins that interacted with these domains, 27 have PY motifs (Figure ); only 9 proteins in the network have a PY motif and did not interact with a WW domain from Rsp5. Consistent with its role as an E3 ubiquitin protein ligase, Rsp5 interacted with several proteins involved in protein modification and turnover, including members of the ubiquitin modification system (for example, Ubi4, Ubc6 and Ubp10), and ubiquitin-like modifications (Rub1). In addition, we observed the known self-interaction between the third WW domain of Rsp5 and the Rsp5 protein on the microarray [45
]. Surprisingly, we did not observe interactions between the Rsp5 WW domain probes and two members of a known Rsp5 complex, Bul1 and Bul2 [46
], both of which are present on our arrays and contain PY motifs. As these proteins are members of a complex, it is possible that accessory proteins needed to mediate the interaction of Rsp5 with Bul1 and Bul2 are not present on the microarray.
A total of 8 proteins in the network have matches to the poly-proline motifs (PPLP and PPPP), and 26 proteins have matches to the PPR motif. Several of these proteins are promiscuous; for example, 2 proteins with poly-proline motifs and 6 proteins with PPR motifs interacted with half or more of the WW domains. This scattered distribution may reflect some intrinsic property of interactions between these ligand classes and WW domains, such as relatively weak affinities between these molecules in the context of microarrays.
The WW domain from Ess1 belongs to the group IV class, which binds phosphorylated ligands. However, because we do not know the phosphorylation states of proteins on the microarrays, we cannot assess the proportion of phosphorylation-dependent interactions within the network. Rpo21, the Pol II subunit containing the carboxy-terminal domain that is bound by Ess1 when phosphorylated, is not present on the microarrays. However, proteins containing WW domains have been proposed to mediate a physical coupling between the transcription and splicing processes in yeast [10
]. Consistent with this association, we observed an interaction between Ess1 and Prp2, a DEAD-box RNA-dependent ATPase required for the first step of mRNA splicing [47
Approximately 43% of the proteins within the network have matches to the canonical ligand motifs known to mediate WW domain interactions. The absence of known motifs in other interacting proteins could be due to any of several reasons. First, isolated WW domains may recognize novel sequence motifs when they are removed from their protein context. Second, they may bind to structural motifs that have yet to be identified at a primary sequence level. Third, other accessory proteins may be needed for WW-containing proteins to recognize their targets.
The lack of known motifs could also be due to more general consequences of using the microarray strategy to identify protein ligands. In a microarray experiment, the concentration of probe protein defines the upper limit of affinity for an interaction. Our probes were applied at low micromolar concentrations, and, therefore, interactions with KD
values higher than this limit would be missed; most of the KD
values measured for WW domain:ligand interactions are in the 10 to 100 μM range [13
]. On the other hand, the concentration of probe may be so high as to recover interactions that are not physiologically relevant. These false-positives could account for spurious interactions with proteins that lack canonical ligand motifs, or have a particular motif but are not bound in vivo
As nearly half of the proteins in the network do not have recognizable WW domain ligand motifs, we searched for novel motifs within the network using motif identification software, including MEME [48
] and a network-based motif sampler [49
]. These approaches did not identify any novel motifs, indicating either that most common motifs have been identified, or that additional parameters such as structural information may be needed to define novel motifs. However, the MEME searches converged on degenerate versions of the PY and LPxY motifs. Many WW domains possess some level of recognition flexibility toward peptide ligands in vitro
, and we asked whether this same versatility was reflected among the proteins within the WW domain network.
Phylogenetic evidence for structural conservation of WW domain ligands
We used a comparative genomics approach to analyze the distribution and conservation of WW domain binding sites. Similar approaches have been used to annotate genomes, to search for conserved functional DNA elements, such as transcription factor binding sites [50
], to discover novel protein interactions [52
], and to delineate receptor-ligand interactions [53
]. Recently, the strategy was used to analyze the yeast SH3 domain interaction network, illustrating that the comparative approach, in combination with protein disorder prediction, was effective in recovering known interactions and predicting novel ones [54
]. Because the peptide ligands bound by WW domains are small, well-defined and sufficient for binding (for example, Pro-Pro-Xaa-Tyr), the search for evolutionarily conserved WW binding sites within protein partners can potentially be reduced to the identification of conserved stretches of amino acid residues.
We compiled genomic sequences for several yeast species in the ascomycete
lineages and searched for orthologs of proteins in our interaction network using the best-hit reciprocal BLAST method [55
]. Of the 207 S. cerevisiae
proteins in the network, 191 have at least one ortholog among the 24 yeast species analyzed. We also analyzed the conservation of the WW domains themselves among yeast lineages (Figure ). The WW domains in Rsp5, Prp40, Ess1, Wwm1, Aus1 and Ypr152c are maintained in all the yeast species. The WW domain in Set2 orthologs is either missing, or is found as one of two classes: the group II/III domain, or, in species closely related to S. cerevisiae
, a meta-WW domain, which lacks the residues defining the group II/III class. The distribution of WW domains among Alg9 orthologs is mainly restricted to species closely related to S. cerevisiae
, whereas that of Ssm4 and Vid30 is only in the S. cerevisiae
Figure 6 Phylogenetic conservation of WW domains among yeast lineages. Radial trees were generated based upon multiple alignments for orthologs culled from 24 yeast species. Solid lines indicate lineages in which the WW domain is maintained in the orthologous (more ...)
These sets of orthologous protein sequences were used to generate multiple sequence alignments, which were examined for the conservation of known primary sequence motifs. In several instances, known WW ligand sequence motifs are conserved among the lineage of interactor orthologs (Figure ; Additional data file 4). Moreover, we found evidence suggesting that WW domains have sufficient recognition malleability to bind structurally similar peptide ligands within the PY (PPxY) and LPxY ligand classes. Both the PPxY and LPxY motifs were found in sets of orthologs as: an invariant sequence; multiple sequences in which the 'x' position varies; or multiple sequences in which the tyrosine is replaced with structurally similar residues (predominantly phenylalanine but in some instances histidine or tryptophan). Although the first two classes were expected, the third class has not been previously observed in a biological context. However, the group I WW domains exhibit recognition flexibility in vitro
. Previously, the specificity of the Yap65 WW domain was assessed using an array of peptides encompassing each single alanine substitution of the peptide ligand, demonstrating that phenylalanine is a functional replacement for tyrosine within the PPxY motif [56
]. Several group I WW domains also exhibit this recognition flexibility [57
]; the structure of a Nedd4 WW domain-PPxY ligand indicated that peptide binding uses a groove that recognizes the N-substituted Pro-Pro sequence, forming a large pocket that accommodates the tyrosyl side chain [16
]. It is possible that phenylalanine side chains are accommodated by this pocket, and that the subtle tyrosine to phenylalanine structural change may be used in biological contexts for the tuning of WW domain-ligand interactions.
Figure 7 Phylogenetic conservation of the WW ligand motifs within yeast proteins. (a) Positions of primary sequence motifs within S. cerevisiae Aat2, Ymr171c, Ylr392c, Prp2, and Yjl084c. (b) Logo representations  of the conserved region within the set of orthologs. (more ...)
We analyzed several conserved motifs in detail (Figure ). Ymr171c, an endosomal protein of unknown function that interacted with the third WW domain from Rsp5, harbors two PPxY motifs that are maintained in nearly all of its 21 orthologs. Aat2 is an aspartate aminotransferase that localizes to peroxisomes during oleate utilization [58
]. It contains a single PPxY motif that is maintained as PPxH and PPxF in several of the orthologs. Ylr392c contains single instances of the PPxY, PPxF and LPxY motifs, each of which is conserved among its three orthologs. Ylr392c interacted with the first and third WW domains of Rsp5, a finding that is supported by its prior identification via AP-MS as a member of an Rsp5 complex [5
]. Yjl084c contains instances of the PPxY, PPxF and LPxY motifs. The PPxY and LPxY motifs are maintained in all 19 orthologs, while the PPxF motif is present in 15 of the orthologs. Yjl084c interacted with the first and third domains of Rsp5, and is known to be phosphorylated by Cdk1 [59
]. Finally, Prp2 is an essential RNA helicase that participates in the early steps of mRNA splicing. Prp2 has two LPxY motifs that are conserved among its ten orthologs. Prp2 was found by five WW domain probes, possibly indicating a reduction in specificity for the LPxY motif.
These motifs may represent structural determinants that are evolutionarily maintained because of a selective pressure applied by their interactions with WW domain-containing proteins. This hypothesis relies on the assumption that the presence of a protein sequence motif (for example, PPxY) is sufficient to mediate an interaction with a WW domain. We tested this assumption by asking whether these putative WW domain recognition determinants are more conserved than similar determinants. For each set of orthologs, we used the S. cerevisiae protein as a reference point and asked to what extent other determinants of a similar form are conserved. For example, both the PPxY and LPxY motifs can be generalized as tripeptides with an intervening residue (that is, X-X-x-X). For each such tripeptide in the S. cerevisiae protein, we determined the proportion of orthologs that maintained the three residues, allowing all substitutions at the 'x' position. We generated histograms of these data, and labeled the bins that contain the putative determinant (for example, PPxY) present in the S. cerevisiae protein (Figure ). In each case except that of Aat2, the putative determinants are among the most highly conserved motifs within the set of orthologs, suggesting that these sequences are being actively maintained. In the Aat2 lineage, PPxY is found as PPxH and PPxF in several of the orthologs, reducing its apparent conservation level. Of the 54 ortholog groups that have instances of the PPxY, PPxF, LPxY or LPxF motifs, we found 27 orthologous protein sets in which the motif is maintained in more than half of the orthologs, suggesting that maintenance of these determinants is common among the proteins found to interact with WW domains (Figure ).
Histograms representing the levels of conservation for the PPxY, PPxF, LPxY and LPxF motifs among 54 orthologous protein sets.
When structural malleability within WW domain ligands was observed, the results were initially disregarded as in vitro
artifacts. Here, we have presented evidence that recognition versatility is sufficiently widespread as to be conserved in several protein lineages from evolutionarily distant yeast species. To address the limits of this conservation, we performed a re-evaluation of a recent study [43
] of human WW domain interactions based on epitope tagging and AP-MS. Several of the co-purifying proteins do not have matches to the canonical sequence motifs that were initially analyzed [43
]. However, we found that many of the human proteins have matches to the PPxF and LPxY motifs, including splicing and transcription factors (for example, PPxF and LPxY in U2AF2, LPxY in CPSF1) (Additional data file 5).
Several WW domain proteins have conserved WW domain binding sites
Searches for primary sequence motifs within the WW domain-interacting orthologs indicated that several of the WW domain-containing proteins themselves have evolutionarily conserved WW domain binding sites (Figure ). A similar observation [60
] was made for Rsp5, which binds peptides harboring the LPxY motif that is found at the carboxyl terminus of Rsp5. Our analysis revealed that Alg9 also has a conserved LPxY motif that in some lineages is coincident with presence of the WW domain, possibly indicating a co-evolving domain and binding site (Figure ). In addition, the Wwm1 and Ssm4 proteins harbor PY motifs (PPxY in Wwm1, PPxF in Ssm4), which are maintained in nearly all of their respective orthologs. We analyzed these proteins for the conservation of S. cerevisiae
protein motifs and found that for Rsp5, Wwm1 and Ssm4, the putative WW domain binding sites are among the most conserved motifs within these proteins (Figure ). The LPxY determinant in Alg9 is less well-conserved, which may indicate that it is not used as a WW domain recognition site.
Figure 9 Co-occurrence of WW domains and WW domain binding sites. (a) Positions of the WW domains (green bars) and conserved primary sequence motifs (PPxY/F in red, LPxY/F in blue) in Wwm1, Rsp5, Alg9 and Ssm4. (b) Radial trees and motif conservation for the ortholog (more ...)
The pattern of conservation for the Wwm1, Rsp5 and Ssm4 proteins is suggestive of two separate types of evolutionary maintenance. The first is self-interaction, as when the WW domains and recognition sites are co-maintained in Wwm1 and Rsp5. We observed an interaction between the third WW domain of Rsp5 and the Rsp5 protein on the microarray, supporting the conservation a WW domain binding site. In our study, Wwm1 was present on the microarrays but did not interact with the Wwm1 WW domain probe. The second type of maintenance is binding of a conserved recognition site to another WW domain-containing protein that is present throughout the lineage. For Ssm4, the WW domain is present only in the S. cerevisiae protein, whereas WW domain binding sites are present in nearly all of the orthologs. As both Ssm4 and Rsp5 are ubiquitin ligases, it is possible that the conserved PPxF site in Ssm4 mediates an interaction with Rsp5; the presence of the WW domain in the S. cerevisiae Ssm4 ortholog may thus reflect a unique functional specialization. In our study, Ssm4 was not present on the microarrays.
The role of Wwm1 in the yeast apoptotic response [27
] may be mediated by its interaction with either itself or other proteins containing WW domains, possibly serving to propagate some signal necessary for regulation of this response. Wwm1 interacted with Pai3, the cytoplasmic inhibitor of yeast saccharopepsin [61
]. As the apoptotic cascade in higher eukaryotes is initiated by a series of proteolytic cleavage events, the Wwm1-Pai3 interaction may point to a similar protease-initiated cascade of signaling events in yeast.
A model for WW domain interaction evolution
Isolated WW domains bind their cognate ligands weakly in vitro
, with KD
s in the 10 to 100 μM range [13
] (Figure ). However, the biological context of many WW domains and their protein ligands likely serves to increase the affinity of these interactions. Two broad classes of binding modes could increase the apparent affinity of interactions (Figure ). One class is represented by proteins that have multiple WW domains and bind ligands with isolated motifs, whereas the other class contains proteins with a single WW domain whose ligands contain multiple binding sites. Both of these situations are frequently observed: Rsp5 and Prp40 in S. cerevisiae
and several human proteins contain multiple WW domains [43
]. Conversely, the S. cerevisiae
Ess1 protein (Pin1 in humans) interacts with several repeats of the phospho-Ser/Thr-Pro motif in the Pol II carboxy-terminal domain [10
]. Coincident WW domain and WW binding sites (Figure ) such as those found in the Rsp5, Ssm4, Alg9 and Wwm1 proteins could influence function by serving as sites for either intra- or intermolecular association. Such associations could provide a mechanism for self-imposed regulation, or could play a more active role by increasing the local concentration of an ancillary functional domain, labeled 'X' in Figure . Analysis of the interactions of the WW domains of human Nedd4 family proteins showed that whereas some proteins were recognized uniquely by a WW domain, others were recognized by multiple WW domains [43
], supporting a model for interaction specificity tuning. WW domains may thus act as scaffolds in the construction of multi-protein complexes by providing a mechanism for the optimization of specificity and affinity for the interactions between WW domains and their protein partners.
A model for the optimization of interactions between WW domains and protein ligands. WW domains are colored green, WW ligand binding motifs are colored red, and auxiliary protein domains are in blue.