Binding of a proline-independent hydrophobic motif by the Candida albicans Rvs167-3 SH3 domain

Src-homology 3 (SH3) domains are small protein-protein interaction modules. While most SH3 domains bind to proline-x-x-proline (PxxP) containing motifs in their binding partners, some SH3 domains recognize motifs other than proline-based sequences. Recently, we showed that the SH3 domain of Candida albicans Rvs167-3 binds peptides enriched in hydrophobic residues and containing a single proline residue (RΦxΦxΦP, where x is any amino acid and Φ is a hydrophobic residue). Here, we demonstrate that the proline in this motif is not required for Rvs167-3 SH3 recognition. Through mutagenesis studies we show that binding of the peptide ligand involves the conserved tryptophan in the canonical PxxP binding pocket as well as residues in the extended n-Src loop of Rvs167-3 SH3. Our studies establish a novel, proline-independent, binding sequence for Rvs167-3 SH3 (RΦxΦxΦ) that is comprised of a positively charged residue (arginine) and three hydrophobic residue

Identification and characterization of Rvs162/Rvs167-3, a novel N-BAR heterodimer in the human fungal pathogen Candida albicans

Membrane reshaping resides at the core of many important cellular processes and amongst its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro. A second, distinct, heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes indicating that C. albicans expresses two functional BAR heterodimers. We used life cell imaging to localize GFP-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly co-localizes with the endocytic marker protein Abp1, we do not observe any co-localization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt or decreased cell wall integrity, phenotypes observed for rvs167Δ/Δ and rvs161Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans: the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have a related but distinct membrane-associated function

Approach used to characterize SH3 domain specificity conservation in four model yeasts.

<p>(<b>A</b>) Overview of the approach we used to characterize the SH3 domain specificity landscape in four yeast species that span an evolutionary distance of some 400 Ma. (<b>B</b>) Overview of all SH3 domain proteins in <i>S</i>. <i>cerevisiae</i> (<i>Sc</i>), <i>A</i>. <i>gossypii</i> (<i>Ag</i>), <i>C</i>. <i>albicans</i> (<i>Ca</i>) and <i>S</i>. <i>pombe</i> (<i>Sp</i>). The dendrogram derived from their full multiple sequence alignment illustrates the diverging sequence conservation of orthologs and paralogs, analogous to the evolutionary distance among the four different yeasts. Alternating colors of red and blue indicate conserved families. Previously non-described <u>S</u>H3 domain <u>c</u>ontaining <u>p</u>roteins (Scp) that could not be confidently assigned to a family are shown in grey.</p

Scanning of homologous binding partner sequences with SPOT-derived PWMs reveals conservation of binding sites.

<p>(<b>A</b>) Sequence scanning of the <i>Sc</i>App1 and <i>Sc</i>Las17 homologs with a PWM of the Myo5 SH3 domains reveals that <i>Sp</i>App1 lost its Myo5 SH3 binding motif. The presence of multiple polyproline motifs in Las17 is conserved across all four yeasts. (<b>B</b>) Sequence scanning of the <i>Sc</i>Abp1 and <i>Sc</i>Gyp5 homologs shows that both <i>Sp</i>Gyp5-1 and SpGyp5-2 lost their Rvs167 SH3 binding motif. All significant hits are indicated by lollipops and colored according to the motif type (Type I, blue; Type II, red; TypeI/II, blue/red; Type III, green). Asterisks indicate previously reported interaction sites.</p

Myo5 and Rvs167 binding validation.

<p>(<b>A</b>) Sepharose-bead bound GST or GST-tagged C-terminal myosin type I tails of <i>Sc</i>Myo5 (984–1219), <i>Sc</i>Myo3 (1010–1271), <i>Ag</i>Myo5 (1084–1292), <i>Ca</i>Myo5 (1004–1316) and <i>Sp</i>Myo1 (1967–1217) were incubated with a total protein extract of <i>S</i>. <i>cerevisiae</i> supplemented with TRITC-labeled actin. The fluorescent halos around the beads (sized 50–150 μm) show the ability of the myosin type I tails of the four different yeast species to recruit active actin polymerization machinery to the beads while the negative control GST does not. Addition of 10 μM Latrunculin A inhibits actin polymerization. (<b>B</b>) Yeast two-hybrid strains co-transformed with the indicated bait and prey constructs were spotted (~10⁴ cells) on minimal plates with histidine (His⁺), without histidine (His⁻), without histidine containing 2.5, 5, or 10 mM 3-amino-1,2,4-triazole (3AT), or without adenine (Ade^–). Weak interactors activate only the <i>HIS3</i> reporter and show growth on His⁻plates, while strong interactors activate both <i>HIS3</i> and <i>ADE2</i> reporters and show growth on His⁻plates containing 3AT or on Ade⁻plates. Note that <i>Ca</i>Rvs167-3 SH3 shows weak self-activation as revealed by growth on His⁻plates in the presence of an empty bait plasmid.</p

Within-family comparisons of specificity profiles highlight a novel diverged specificity class for <i>Ca</i>Rvs167-3.

<p>(<b>A</b>) Separately clustered heat maps of the Rvs167 and Myo5 families show that both families have a high degree of binding profile conservation among orthologs, with the exception of <i>Ca</i>Rvs167-3, whose binding profile does not correlate with any of the Rvs167 orthologs. (<b>B</b>) Specificity logos built from manual alignments of the top 10 binding peptides show that, with the exception of <i>Sp</i>Rv167, all Rvs167 binding peptides could be aligned as Type I and II profiles (left). The <i>Ca</i>Rvs167-3 binding profile forms a distinct Type I-like (Type I*) class, characterized by the presence of a hydrophobic residue instead of the first proline. All Myo5 ortholog binding profiles show a clear disposition for a poly-proline motif, devoid of charged residues (right).</p

Evolution of the SH3 domain specificity landscape in yeasts

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.LS, BW, FH and BD received funding from the European Union (Marie Curie Research Training Network Penelope; MRTN-CT-2006-036076)