A genome-wide study of PDZ-domain interactions in C. elegans reveals a high frequency of non-canonical binding
- Nicolas Lenfant†1,
- Jolanta Polanowska†1, 3,
- Sophie Bamps1, 2,
- Shizue Omi1, 3,
- Jean-Paul Borg1 and
- Jérôme Reboul1, 3Email author
© Lenfant et al; licensee BioMed Central Ltd. 2010
Received: 15 September 2010
Accepted: 26 November 2010
Published: 26 November 2010
Proteins may evolve through the recruitment and modification of discrete domains, and in many cases, protein action can be dissected at the domain level. PDZ domains are found in many important structural and signaling complexes, and are generally thought to interact with their protein partners through a C-terminal consensus sequence. We undertook a comprehensive search for protein partners of all individual PDZ domains in C. elegans to characterize their function and mode of interaction.
Coupling high-throughput yeast two-hybrid screens with extensive validation by co-affinity purification, we defined a domain-orientated interactome map. This integrates PDZ domain proteins in numerous cell-signaling pathways and shows that PDZ domain proteins are implicated in an unexpectedly wide range of cellular processes. Importantly, we uncovered a high frequency of non-canonical interactions, not involving the C-terminus of the protein partner, which were directly confirmed in most cases. We completed our study with the generation of a yeast array representing the entire set of PDZ domains from C. elegans and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest.
We provide an extensive domain-centered dataset, together with a clone resource, that will help future functional study of PDZ domains. Through this unbiased approach, we revealed frequent non-canonical interactions between PDZ domains and their protein partners that will require a re-evaluation of this domain's molecular function.
[The protein interactions from this publication have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct (PMID: 19850723) and assigned the identifier IM-14654]
Because of its biological importance, the PDZ (PSD-95, Discs-large, ZO-1) domain has been intensively studied at the structural and functional level. Proteins containing PDZ domains frequently serve as molecular scaffolds, which assemble signaling complexes needed for efficient and specific signal transduction at defined sub-cellular sites, such as at polarized epithelial cell junctions, or synapses in neurons [1–3]. Early work indicated a preferential interaction between PDZ domains and the C-terminal amino acids of target proteins . In some cases, removal of the 3 C-terminal residues of the partner protein abrogates interaction with the PDZ domain . Much subsequent effort has been put into bioinformatic studies and small- and large-scale screens to refine the exact sequence of this presumed C-terminal motif [5–10], leading to several consensus sequences, with different degrees of refinement (e.g. Additional file 1; [1, 10–12]). Individual proteins can contain multiple PDZ domains. For example, the human multiple PDZ domain protein (MPDZ) has 13. When their interactions with other proteins have been dissected, the different PDZ domains of a single protein often have been found to have distinct binding partners (see for example the Uniprot entry for MPDZ ). PDZ domain proteins have also been used in the context of large-scale searches for protein partners. For example, global interactome studies with C. elegans proteins assayed the interactions of 25 of the nematode's 62 PDZ domain proteins. Although these 25 proteins were found to be involved in 218 interactions, whether the different PDZ domains played a direct role was not addressed [14, 15]. No comprehensive, proteome-wide screen using all PDZ domains, however, has been reported for any organism.
Here, we describe the characterization and cloning of every single one of the 93 PDZ domains from C. elegans. We generated a versatile resource, with each domain in the Gateway system, allowing facile transfer to different expression systems. As an example, we made a yeast array of the 93 PDZ domains and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest. In addition, from a separate yeast two-hybrid (Y2H) screen, we identified more than 650 potential partners for these domains. A large number of these interactions were independently validated using a co-immunoprecipitation approach. An analysis of these interactors implicates PDZ domains in a broad range of cellular functions. Unexpectedly, many of the interactions did not involve a C-terminal consensus sequence, suggesting that PDZ domains frequently bind their partners in a hitherto uncharacterized mode.
An interactome map for PDZ domains
Proportions of C-terminal consensus classes in interacting proteins.
consensus class 1
consensus class 2
consensus class 3
total non consensus
n = 447
n = 227
C. elegans proteome
n = 20186
consensus class 1
consensus class 2
consensus class 3
total non consensus
n = 317 nr
n = 178 nr
C. elegans proteome
n = 20186
Characteristics of PDZ domain interacting proteins
Frequent use of non-consensus binding confirmed by co-IP
A Y2H array as a tool to probe PDZ domain binding
We constructed a comprehensive, proteome-wide interaction map for all the PDZ domains from C. elegans. Importantly, for a substantial proportion of the interactions, we were able to obtain independent biochemical confirmation. The interactions we characterized covered a broad range of putative biological functions, reflecting the ubiquitous involvement of PDZ-domain proteins in cellular physiology. Although a number of PDZ-domain proteins have been functionally characterized in great details, in very few cases has a role for an individual PDZ-domain been identified. We did find a small group of interactions involving PDZ-domains protein for which there was prior experimental evidence, such as those involving LET-23 and LIN-7, and PAR-3 and PKC-3 [28, 31]. Further, we were able to provide a molecular basis for certain previously characterized genetic interactions (e.g. between the polarity gene par-6 and the RhoGAP pac- 1 ). There were many additional interactions that could merit directed study, such as that between PTEN/DAF-18 and Dishevelled/DSH-1, two proteins that function respectively in the PTEN/AKT and WNT pathway, and the multipartite interaction between LIN-7 and CSC-1, and LIN-10 with CSC-1 and ICP-1. The LIN-2/LIN-7/LIN-10 complex is known for its role in basolateral targeting of the LET-23 receptor. CSC-1 and ICP-1 are orthologs of Borealin and Incenp two components of the vertebrate chromosomal passenger complex (CPC). We confirmed CSC-1's interactions and the interactions between the two PDZ domains of LIN-10 with ICP-1 using a biochemical approach (JP, unpublished results). This raises the possibility of an unsuspected functional link between these two protein complexes, and is a good example of the hypotheses that can be generated through global analyses.
As a last example, both via our global screen and using the PDZ-domain array, we detected an interaction between MIG-5 and PRY-1. Previous studies had mapped the interaction between PRY-1 and MIG-5 to the N-terminal half of MIG-5 , which does contain the protein's single PDZ domain. The C-terminus of PRY-1 (IAAELR) does not contain a consensus PDZ-binding motif. This therefore represents a clear example of a functionally validated protein-protein interaction that we have shown to involve a non-canonical PDZ domain interaction. Indeed, more than half of the interactions did not involve the previously defined PDZ-domain binding C-terminal motifs. By aligning and analyzing our set of PDZ-interacting proteins, we were unable to identify a clear internal motif that could be uniquely responsible for PDZ domain binding. Nevertheless, this global study clearly indicates that non-consensus binding is a much more frequent phenomenon than previously suspected. Extensive future functional studies will be needed to validate all the individual internal PDZ domains interactions described here, but it is important to note that in certain isolated cases, this unconventional mode of binding has been demonstrated [32–42].
It is clear that global Y2H screens only reveal a fraction of potential protein-protein interactions . Among other factors, this is due to cDNA representation in non-normalized libraries. This was one motivation for generating an array that allows direct Y2H assay of any protein or peptide of interest against a complete set of PDZ domains. Coupled with the collection of PDZ domain sequences in the Gateway entry vector, allowing facile transfer to vectors for RNAi, or protein expression, the array, which is available as a community resource, will allow comprehensive functional analyses of all PDZ domains in C. elegans.
By conducting a comprehensive, domain-centered interactome study, we have clearly illustrated at the genome scale the degree of promiscuity and discrimination that governs interactions between individual PDZ domains and their protein partners. This approach also revealed that PDZ domains frequently interact in a non-canonical fashion. This broadens our understanding of PDZ domains and should guide future functional studies.
PDZ domain identifications in C. elegans proteome
Domain boundaries where obtained by cross searching Wormbase WS150  and SMART version 4.0 (genomic mode) (http://smart.embl-heidelberg.de) . Each domain was extended on each side with a 10 amino acid tail from the original protein to ensure the integrity of the structure of the domain. In some cases size of these tails had to be slightly modified according to the position of the PDZ in protein (extreme end or start) or to ensure a correct amplification.
PDZ domain cloning
Primers, containing Gateway B1 and B2 recombination tails, were designed using the OSP program as described [46, 47] including a stop codon before the B2 tail (see Additional file 2: Supplemental Table S1). DNA fragments encoding each PDZ domain where amplified by polymerase chain reaction (Platinum HIFI polymerase, Invitrogen) and cloned into pDONR201 Entry vector using the Gateway recombinational cloning system as described [17, 18]. PDZ Entry clones were sequence verified using P201DONRF primer 5'-TCGCGTTAACGCTAGCATGGATCTC and then used in a Gateway LR recombination reaction to transfer the DNA coding for the PDZ domain into the yeast expression vector pPC97-Dest as described .
Transformation of pDB-ORFs into yeast cells and removal of auto-activators
DB-ORF plasmids were transformed into yeast strain MaV203 using standard transformation protocols . Auto-activators were identified by testing the activation of GAL1::HIS3 on minimal medium lacking leucine and histidine but containing 20 mM 3-amino-1,2,4-triazole (3-AT) in the absence of any AD-containing vector.
Identification of interacting protein pairs
Bait strains containing a single pDB-PDZ were individually transformed with the C. elegans AD-wrmcDNA and AD-ORFeome1.0 libraries  as described . A minimum of 1 × 106 colonies were screened for each bait strain tested with the AD-wrmcDNA library and a minimum of 1.5 × 105 colonies were screened for each bait strain tested with the AD-ORFeome library. After 4 to 5 days at 30°C, single 3-AT resistant colonies were picked on synthetic complete medium lacking leucine, tryptophan, and histidine and containing 20 mM 3-AT (SC, Leu-, Trp-, His-, 20 mM 3-AT) and then rearrayed on fresh SC, Leu-, Trp-, His-, 20 mM 3-AT plates.
Colonies able to grow on SC, Leu-, Trp-, His-, 20 mM 3-AT plates were tested for expression of three Y2H reporter genes (GAL1::HIS3, GAL1::lacZ, and SPAL10::URA3, as described .
ORF insert sequencing
To prepare DNA for PCR, yeast colonies were re-suspended in 15 μl lysis buffer (50 units zymolase in 0.1 M Na-Phosphate buffer pH 7.4) using toothpicks, and lysed by incubating for 10 min. at 37°C and 10 min. at 95°C. For each PCR, 0.3 μl of lysis mix was used. AD inserts were amplified using primers 5'-CGCGTTTGGAATCACTACAGGG and 5'-GGAGACTTGACCAAACCTCTGGCG (AD and TERM respectively). DB inserts were amplified using primers 5'-GGCTTCAGTGGAGACTGATATGCCTC (DB) and TERM. PCR products were sequenced using the AD or DB primers.
Sequence trace analysis
Colonies showing an activation of at least two of the three Y2H reporter genes were PCR amplified, as described above. PCR products showing a single band on ethidium bromide gel were sent for sequencing. The quality of the sequence obtained was determined as described  by moving a sliding window of 10 base pairs along the sequence to define the portion that has an average PHRED score of 20 or higher [49, 50]. Sequences for which less than 15% of their length met this criterion were discarded. A nucleotide BLAST  search was performed against Wormpep150  to determine the identity of the clone. Finally, the reading frame was obtained by local alignment of the 3' end of the Gal4 AD encoding sequence with the 5' end of the prey encoding sequence. A translation according to this reading frame was used to perform a protein BLAST search against Wormpep150. If the nucleotide and protein BLAST agreed, the prey encoding sequence was considered "In Frame", otherwise it was designated as "Out of Frame" and discarded.
Gap repair was used to retest all Y2H interactions as described . When an interaction failed to be re-confirmed it was discarded from the dataset.
Construction and screening of a comprehensive PDZ domain Y2H array
All PDZ domains were transferred into AD vector (pACT2) by Gateway recombinational cloning and transfected into the haploid Y187 yeast strain (MAT α, ura3-52, his3-200, ade2-101, leu2-3, 112, gal4 Δ, met-, gal80 Δ, MEL1, URA3::GAL1UAS -GAL1TATA-lacZ). Individual ORFs of proteins of interest were cloned into DB vector (pGBT9) by Gateway recombinational cloning and the resulting constructs transformed into haploid AH109 yeast strain (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4 Δ, gal80 Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1). Interactions between each PDZ and a given ORF was tested through mating of the two yeast strains. Phenotypic testing evaluated growth of diploid cells on selective medium (Leu-, Trp-, His-, 2 mM 3-AT), which is dependent in part upon the expression of the GAL1::HIS3 selective marker gene.
Co-IP verification of Y2H interacting pairs
To test interactions identified in the AD-wrmcDNA library Y2H screen using co-IP (Figure 3, Additional file 5: Supplemental Table S10), the full length ORF coding for the target protein identified was amplified from the AD-wrmcDNA library, Gateway cloned into the pDONR201 Entry vector and transferred using the LR reaction into the expression vector pDEST-CMV-MYC which contains a MYC tag upstream of the B1 recombination site. For each fragment the endogenous Stop codon was preserved before the B2 recombinational tail (the endogenous C-terminus of the corresponding protein fragment was preserved).
To test interactions identified in the AD-wrmcDNA or AD-ORFeome screens, or both (see Additional file 6, Additional file 5: Supplemental Table S11), but using a B2-tailed construct for each PDZ-domain interacting protein, clones corresponding to the full length protein in the pDONR201 entry vector were retrieved from the C. elegans ORFeome collection . Because of the nature of the constructs used in the ORFeome, LR-transfer of the ORF into the pDEST-CMV-MYC expression vector produced a protein ending with the C-terminal amino-acid sequence PAFLYKVVIIHSSMHLEGPIL (B2 encoding tail + 13 aa on pDEST-CMV-MYC before Stop codon).
Internal interactions (Figure 4, Additional file 7, Additional file 5: Supplemental Table S12 and S13) were tested by co-IP using 74 interactions for which the pair of PDZ/interacting proteins was found multiple times through the screening process of the AD-wrmcDNA library but that had no C-terminal consensus PDZ binding motif. These interactions corresponded to 59 different interacting proteins. To ensure a maximum reproducibility with the Y2H interactions, sequence data from the Y2H screen was used to define the smallest cDNA fragment identified among all clones obtained for each interaction in the Y2H screen (designated as the minimal interacting region, or MIR). For each fragment the codons encoding for the last three amino acid were removed from the primers and replaced by a Stop codon, giving rise after PCR amplification, Gateway cloning into the pDONR201 Entry vector and LR-transfer into the pDEST-CMV-MYC expression vector, to a cloned fragment encoding a protein lacking the last three amino acids. This was done to ensure that proteins could not interact by their native C-terminus, so that a positive result would provide support for an internal mode of interaction. When a PDZ-interacting protein was present in multiple pairs of interactions the smallest cDNA fragment of all pairs was used to test all interactions.
For all Co-IP experiments in this study, DNA encoding each PDZ domains tested was transferred from the pDONR201 Entry vector to the pDEST-CMV-3xHA expression vector containing the 3 × HA sequence upstream of the B1 recombination site.
Plasmids pDEST-CMV-3xHA and pDEST-CMV-MYC expressing their fusion proteins from the CMV promoters were transfected into 293T cells using Fugen 6 transfection reagent according to the manufacturers instructions (Roche). Cells were cultured for 48 hours in DMEM medium, and lysed in 0.1% NP-40 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and complete protease inhibitors and phosphatase inhibitors (Thermo Scientific)). Lysates were cleared by centrifugation at 14,000 × g and subjected to co-immunoprecipitations of protein complexes using anti-HA (clone12CA5) sepharose beads. Purified complexes and control lysate (10 μg of total protein) samples were separated on Nu-PAGE Bis-Tris 4-12% gels (Invitrogen), and MYC and HA tagged proteins were detected using standard immunoblotting techniques. Antibodies used were mouse monoclonal anti-MYC (clone 9E10, Sigma) and monoclonal anti-HA (clone HA.11, Covance).
The Textpresso database  was used to search for interactions were both bait and prey proteins had public alphanumeric gene names. GO terms attributes were retrieved from Wormbase. Note that in C. elegans most attributes are currently inferred from electronic annotation (IEA).
We thank Jonathan Ewbank. This work was supported by an institutional grant from INSERM, the INSERM-AVENIR program and Association pour la Recherche sur le Cancer. N.L. was funded by INSERM-Region PACA and Association pour la Recherche sur le Cancer PhD fellowship. S.B. was a recipient of INSERM Poste Vert fellowship.
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