Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins

Abstract

Background: The WW domain is found in a large number of eukaryotic proteins implicated in a variety of cellular processes. WW domains bind proline-rich protein and peptide ligands, but the protein interaction partners of many WW domain-containing proteins in Saccharomyces cerevisiae are largely unknown.

Results: We used protein microarray technology to generate a protein interaction map for 12 of the 13 WW domains present in proteins of the yeast S. cerevisiae. We observed 587 interactions between these 12 domains and 207 proteins, most of which have not previously been described. We analyzed the representation of functional annotations within the network, identifying enrichments for proteins with peroxisomal localization, as well as for proteins involved in protein turnover and cofactor biosynthesis. We compared orthologs of the interacting proteins to identify conserved motifs known to mediate WW domain interactions, and found substantial evidence for the structural conservation of such binding motifs throughout the yeast lineages. The comparative approach also revealed that several of the WW domain-containing proteins themselves have evolutionarily conserved WW domain binding sites, suggesting a functional role for inter- or intramolecular association between proteins that harbor WW domains. On the basis of these results, we propose a model for the tuning of interactions between WW domains and their protein interaction partners.

Conclusion: Protein microarrays provide an appealing alternative to existing techniques for the construction of protein interaction networks. Here we built a network composed of WW domain-protein interactions that illuminates novel features of WW domain-containing proteins and their protein interaction partners.

Figure 1

Motifs in yeast WW domain proteins and WW sequence alignment. (a) Ten yeast proteins contain a total of thirteen WW domains. (b) Multiple sequence alignment of the 13 WW domains. The domains from Rsp5 and Prp40 are named corresponding to their occurrence from amino to carboxyl terminus. Conservation of the tryptophan residue at position 13 and the proline residue at position 39, as well as partial conservation of the tryptophan at position 36 define the WW domain (filled blue boxes). The sequences shown were purified as fusions to either MBP or GST. Residues boxed in red residues indicate the sequence determinants that put the WW domains into three different classes: groups I, II/III and IV [13]. Six of the WW domains do not conform to any of the classifications.

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 antibody (right middle panel), and biotinylation of fusion proteins observed by binding of HRP-conjugated streptavidin (bottom panels) are shown.

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 (Ubc6 and Oye3) had signals above background. Control proteins (dye-labeled and biotinylated proteins) are indicated. (b) Interactions involving the WW domains from Rsp5. A total of 124 proteins were identified using the WW domains from Rsp5. Functional annotations are superimposed on the network using filled circles and outlines.

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 protein interactors; power law fits to data sets including (black line) and excluding (red line) WW domain probe are shown.

Figure 5

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.

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 proteins, whereas dashed lines indicate those proteins in which the WW domain is not present. In the Set2 ortholog group, the WW domains highlighted in gray are most similar to the meta-WW domain in S. cerevisiae, whereas in the other lineages the WW domain conforms to the group II/III classification. Organism abbreviations are Saccharomyces cerevisiae (Sc),Candida guilliermondii (Cgui),Candida glabrata (Cgla),Chaetomium globosum (Cglo),Kluyveromyces waltii (Kw),Kluyveromyces lactis (Kl),Yarrowia lipolytica (Yl),Candida lusitaniae (Cl),Debaryomyces hansenii (Dh),Schizosaccharomyces pombe (Sp),Pneumocystis carinii (Pc),Fusarium graminearum (Fg),Magnaporthe grisea (Mg),Neurospora crassa (Nc),Podospora anserina (Pa),Aspergillus fumigatus (Af),Aspergillus nidulans (An),Ashbya gosypii (Ag),Histoplasma capsulatum (Hc),Coccidioides immitis (Ci), Ustilago maydis (Um),Cryptococcus neoformans (Cn),Coprinus cinereus (Cc),and Rhizopus oryzae (Ro).

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 [68] of the conserved region within the set of orthologs. The number of orthologs in each set is indicated. Gray dashed boxes highlight the conserved motifs; numbers indicate the position of the motif within the S. cerevisiae protein. Histograms represent the level of conservation of all S. cerevisiae X-X-x-X sequence determinants within the set of orthologs. Colored circles mark the bins that contain the PPxY, PPxF and LPxY motifs.

Figure 8

Histograms representing the levels of conservation for the PPxY, PPxF, LPxY and LPxF motifs among 54 orthologous protein sets.

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 groups of each protein. Organism abbreviations are Saccharomyces cerevisiae (Sc),Candida guilliermondii (Cgui),Candida glabrata (Cgla),Chaetomium globosum (Cglo),Kluyveromyces waltii (Kw),Kluyveromyces lactis (Kl),Yarrowia lipolytica (Yl),Candida lusitaniae (Cl),Debaryomyces hansenii (Dh),Schizosaccharomyces pombe (Sp),Pneumocystis carinii (Pc),Fusarium graminearum (Fg),Magnaporthe grisea (Mg),Neurospora crassa (Nc),Podospora anserina (Pa),Aspergillus fumigatus (Af),Aspergillus nidulans (An),Ashbya gosypii (Ag),Histoplasma capsulatum (Hc),Coccidioides immitis (Ci), Ustilago maydis (Um),Cryptococcus neoformans (Cn),Coprinus cinereus (Cc),and Rhizopus oryzae (Ro). Solid branches indicate lineages in which the WW domain is present; dashed lines indicate the absence of a WW domain. Colored branches indicate lineage in which a motif is present; lineages in gray lack the motif. The histograms represent the relative conservation of S. cerevisiae motifs of the form X-X-x-X among the orthologs. Red and blue dots indicate the bins that contain the highlighted motifs.

Figure 10

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.