PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes.

The multi-domain scaffolding protein NHERF1 modulates the assembly and intracellular trafficking of various transmembrane receptors and ion-transport proteins. The two PDZ (postsynaptic density 95/disk large/zonula occluden 1) domains of NHERF1 possess very different ligand-binding capabilities: PDZ1 recognizes a variety of membrane proteins with high affinity, while PDZ2 only binds limited number of target proteins. Here using NMR, we have determined the structural and dynamic mechanisms that differentiate the binding affinities of the two PDZ domains, for the type 1 PDZ-binding motif (QDTRL) in the carboxyl terminus of cystic fibrosis transmembrane regulator. Similar to PDZ2, we have identified a helix-loop-helix subdomain coupled to the canonical PDZ1 domain. The extended PDZ1 domain is highly flexible with correlated backbone motions on fast and slow timescales, while the extended PDZ2 domain is relatively rigid. The malleability of the extended PDZ1 structure facilitates the transmission of conformational changes at the ligand-binding site to the remote helix-loop-helix extension. By contrast, ligand binding has only modest effects on the conformation and dynamics of the extended PDZ2 domain. The study shows that ligand-induced structural and dynamic changes coupled with sequence variation at the putative PDZ binding site dictate ligand selectivity and binding affinity of the two PDZ domains of NHERF1.

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 μM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.

Intercellular adhesion complexes must withstand mechanical forces to maintain tissue cohesion, while also retaining the capacity for dynamic remodeling during tissue morphogenesis and repair. Most cell-cell adhesion complexes contain at least one PSD95/Dlg/ZO-1 (PDZ) domain situated between the adhesion molecule and the actin cytoskeleton. However, PDZ-mediated interactions are characteristically nonspecific, weak, and transient, with several binding partners per PDZ domain, micromolar dissociation constants, and bond lifetimes of seconds or less. Here, we demonstrate that the bonds between the PDZ domain of the cytoskeletal adaptor protein afadin and the intracellular domains of the adhesion molecules nectin-1 and JAM-A form molecular catch bonds that reinforce in response to mechanical load. In contrast, the bond between the PDZ3-SH3-GUK (PSG) domain of the cytoskeletal adaptor ZO-1 and the JAM-A intracellular domain becomes dramatically weaker in response to ∼2 pN of load, the amount generated by single molecules of the cytoskeletal motor protein myosin II. These results suggest that PDZ domains can serve as force-responsive mechanical anchors at cell-cell adhesion complexes, and that mechanical load can enhance the selectivity of PDZ-peptide interactions. These results suggest that PDZ mechanosensitivity may help to generate the intricate molecular organization of cell-cell junctions and allow junctional complexes to dynamically remodel in response to mechanical load.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

Protein-protein recognition, frequently mediated by members of large families of interaction domains, is one of the cornerstones of biological function. Here, we present a computational, structure-based method to predict the sequence space of peptides recognized by PDZ domains, one of the largest families of recognition proteins. As a test set, we use a considerable amount of recent phage display data that describe the peptide recognition preferences for 169 naturally occurring and engineered PDZ domains. For both wild-type PDZ domains and single point mutants, we find that 70-80% of the most frequently observed amino acids by phage display are predicted within the top five ranked amino acids. Phage display frequently identified recognition preferences for amino acids different from those present in the original crystal structure. Notably, in about half of these cases, our algorithm correctly captures these preferences, indicating that it can predict mutations that increase binding affinity relative to the starting structure. We also find that we can computationally recapitulate specificity changes upon mutation, a key test for successful forward design of protein-protein interface specificity. Across all evaluated data sets, we find that incorporation backbone sampling improves accuracy substantially, irrespective of using a crystal or NMR structure as the starting conformation. Finally, we report successful prediction of several amino acid specificity changes from blind tests in the DREAM4 peptide recognition domain specificity prediction challenge. Because the foundational methods developed here are structure based, these results suggest that the approach can be more generally applied to specificity prediction and redesign of other protein-protein interfaces that have structural information but lack phage display data.

The PSD-95/Dlg-A/ZO-1 (PDZ) domain is highly expanded, diversified, and well distributed across metazoa where it assembles diverse signaling components by virtue of interactions with other proteins in a sequence-specific manner. In contrast, in the microbial world they are reported to be involved in protein quality control during stress response. The distribution, functions, and origins of PDZ domain-containing proteins in the prokaryotic organisms remain largely unexplored. We analyzed 7,852 PDZ domain-containing proteins in 1,474 microbial genomes in this context. PDZ domain-containing proteins from planctomycetes, myxobacteria, and other eubacteria occupying terrestrial and aquatic niches are found to be in multiple copies within their genomes. Over 93% of the 7,852 PDZ domain-containing proteins were classified into 12 families including six novel families based on additional structural and functional domains present in these proteins. The higher PDZ domain encoding capacity of the investigated organisms was observed to be associated with adaptation to the ecological niche where multicellular life might have originated and flourished. Predicted subcellular localization of PDZ domain-containing proteins and their genomic context argue in favor of crucial roles in translation and membrane remodeling during stress response. Based on rigorous sequence, structure, and phylogenetic analyses, we propose that the highly diverse PDZ domain of the uncharacterized Fe-S oxidoreductase superfamily, exclusively found in gladobacteria and several anaerobes and acetogens, might represent the most ancient form among all the existing PDZ domains.

Reagents that target protein-protein interactions to rewire signaling are of great relevance in biological research. Computational protein design may offer a means of creating such reagents on demand, but methods for encoding targeting selectivity are sorely needed. This is especially challenging when targeting interactions with ubiquitous recognition modules--for example, PDZ domains, which bind C-terminal sequences of partner proteins. Here we consider the problem of designing selective PDZ inhibitor peptides in the context of an oncogenic signaling pathway, in which two PDZ domains (NHERF-2 PDZ2-N2P2 and MAGI-3 PDZ6-M3P6) compete for a receptor C-terminus to differentially modulate oncogenic activities. Because N2P2 has been shown to increase tumorigenicity and M3P6 to decreases it, we sought to design peptides that inhibit N2P2 without affecting M3P6. We developed a structure-based computational design framework that models peptide flexibility in binding yet is efficient enough to rapidly analyze tradeoffs between affinity and selectivity. Designed peptides showed low-micromolar inhibition constants for N2P2 and no detectable M3P6 binding. Peptides designed for reverse discrimination bound M3P6 tighter than N2P2, further testing our technology. Experimental and computational analysis of selectivity determinants revealed significant indirect energetic coupling in the binding site. Successful discrimination between N2P2 and M3P6, despite their overlapping binding preferences, is highly encouraging for computational approaches to selective PDZ targeting, especially because design relied on a homology model of M3P6. Still, we demonstrate specific deficiencies of structural modeling that must be addressed to enable truly robust design. The presented framework is general and can be applied in many scenarios to engineer selective targeting.

Protein-peptide interactions form an important subset of the total protein interaction network in the cell and play key roles in signaling and regulatory networks, and in major biological processes like cellular localization, protein degradation, and immune response. In this work, we have described the LMDIPred web server, an online resource for generalized prediction of linear peptide sequences that may bind to three most prevalent and well-studied peptide recognition modules (PRMs)-SH3, WW and PDZ. We have developed support vector machine (SVM)-based prediction models that achieved maximum Matthews Correlation Coefficient (MCC) of 0.85 with an accuracy of 94.55% for SH3, MCC of 0.90 with an accuracy of 95.82% for WW, and MCC of 0.83 with an accuracy of 92.29% for PDZ binding peptides. LMDIPred output combines predictions from these SVM models with predictions using Position-Specific Scoring Matrices (PSSMs) and string-matching methods using known domain-binding motif instances and regular expressions. All of these methods were evaluated using a five-fold cross-validation technique on both balanced and unbalanced datasets, and also validated on independent datasets. LMDIPred aims to provide a preliminary bioinformatics platform for sequence-based prediction of probable binding sites for SH3, WW or PDZ domains.

Metastatic cells are highly plastic for differential expression of tumor phenotype hallmarks and metastatic organotropism. The signaling proteins orchestrating the shift of one cell phenotype and organ pattern to another are little known. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a molecular pathway organizer, PDZ-domain protein that recruits membrane, cytoplasmic, and cytoskeletal signaling proteins into functional complexes. To gain insight into the role of NHERF1 in metastatic progression, we stably transfected a metastatic breast cell line, MDA-MB-231, with an empty vector, with wild-type NHERF1, or with NHERF1 mutated in either the PDZ1- or PDZ2-binding domains to block their binding activities. We observed that NHERF1 differentially regulates the expression of two phenotypic programs through its PDZ domains, and these programs form the mechanistic basis for metastatic organotropism. The PDZ2 domain promotes visceral metastases via increased invadopodia-dependent invasion and anchorage-independent growth, as well as by inhibition of apoptosis, whereas the PDZ1 domain promotes bone metastases by stimulating podosome nucleation, motility, neoangiogenesis, vasculogenic mimicry, and osteoclastogenesis in the absence of increased growth or invasion. Collectively, these findings identify NHERF1 as an important signaling nexus for coordinating cell structure with metastatic behavior and identifies the "mesenchymal-to-vasculogenic" phenotypic transition as an essential step in metastatic progression.

PDZ domain interactions are involved in signaling and trafficking pathways that coordinate crucial cellular processes. Alignment-based PDZ binding motifs identify the few most favorable residues at certain positions along the peptide backbone. However, sequences that bind the CAL (CFTR-associated ligand) PDZ domain reveal only a degenerate motif that overpredicts the true number of high-affinity interactors. Here, we combine extended peptide-array motif analysis with biochemical techniques to show that non-motif "modulator" residues influence CAL binding. The crystallographic structures of 13 CAL:peptide complexes reveal defined, but accommodating stereochemical environments at non-motif positions, which are reflected in modulator preferences uncovered by multisequence substitutional arrays. These preferences facilitate the identification of high-affinity CAL binding sequences and differentially affect CAL and NHERF PDZ binding. As a result, they also help determine the specificity of a PDZ domain network that regulates the trafficking of CFTR at the apical membrane.

A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved β₂-β₃ loop and an extra-domain helix. Using the CRIPT peptide as a prototypical ligand, our simulations suggest that a network of salt-bridges between the ligand and this loop is necessary for binding. These contacts interconvert between each other on a time scale of a few tens of nanoseconds, making them elusive to X-ray crystallography. The loop is stabilized by an extra-domain helix. The latter influences the global dynamics of the domain, considerably increasing binding affinity. We found that two key contacts between the helix and the domain, one involving the β₂-β₃ loop, provide an atomistic interpretation of the increased affinity. Our analysis indicates that both extra-domain segments and loosely conserved regions play critical roles in PDZ binding affinity and specificity.

Synapse-associated protein 102 (SAP102) and postsynaptic density-95 (PSD-95) bind to NMDA receptors through PDZ domains and cluster at excitatory postsynaptic sites called postsynaptic densities (PSD). We previously reported that PSD-95 containing mutated PDZ domains incapable of ligand binding clustered at synaptic sites with reduced efficiency. Here, we compared the synaptic clustering of the same series of full-length SAP102 mutants in hippocampal neurons. Unexpectedly, ligand-binding deficient mutant SAP102 showed more efficient synaptic localization than wild-type SAP102. Further, when SAP102-PDZ mutants were co-expressed with either the GluN2A or GluN2B NMDA receptor subunit, both subunits showed decreased synaptic clustering, although the mutants were efficiently targeted to the synapses. This finding suggests that direct binding of NMDA receptors with SAP102 is involved in the efficient targeting of NMDA receptors to the synapses, whereas ligand binding of the PDZ domains is not essential for the synaptic clustering of SAP102.

ClpX, a molecular chaperone and the regulatory subunit of the ClpXP protease, is shown to contain tandem modular domains that bind to the C-terminal sequences of target proteins in a manner that parallels functional specificity. Nuclear magnetic resonance studies show that these C-terminal sequences are displayed as disordered peptides on the surface of otherwise folded proteins. The ClpX substrate-binding domains are homologous to sequences in other Clp/Hsp100 proteins and are related more distantly to PDZ domains, which also mediate C-terminal specific protein-protein interactions. Conservation of these binding domains indicates that the mode of substrate recognition characterized here for ClpX will be a conserved feature among Clp/Hsp100 family members and a distinguishing characteristic between this chaperone family and the Hsp70 chaperones.

Human protein tyrosine phosphatase non-receptor type 4 (PTPN4) has been shown to prevent cell death. The active form of human PTPN4 consists of two globular domains, a PDZ (PSD-95/Dlg/ZO-1) domain and a phosphatase domain, tethered by a flexible linker. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We previously demonstrated that the PDZ domain inhibits the phosphatase activity of PTPN4 and that the mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We demonstrate here that the linker connecting the PDZ domain and the phosphatase domain is involved in the regulation of the phosphatase activity in both PDZ-related inhibition and PDZ ligand-related activation events. We combined bioinformatics and kinetic studies to decipher the role of the linker in the PTPN4 activity. By comparing orthologous sequences, we identified a conserved patch of hydrophobic residues in the linker. We showed that mutations in this patch affect the regulation of the PTPN4 bidomain indicating that the PDZ-PDZ ligand regulation of PTPN4 is a linker-mediated mechanism. However, the mutations do not alter the binding of the PDZ ligand. This study strengthens the notion that inter-domain linker can be of functional importance in enzyme regulation of large multi-domain proteins.

Dynamic regulation of synaptic AMPA receptor localization underlies certain forms of synaptic plasticity and researchers are just beginning to identify molecules that may play a role in the synaptic delivery of glutamate receptors. One candidate is mLin-10, the mammalian homolog of the C. elegans receptor targeting protein LIN-10. Here, we investigated the role of mLin-10 in glutamate receptor trafficking. Cellular localization studies, in both whole brain and cultured neurons, revealed that mLin-10 is enriched in the trans-Golgi network and present in dendrites and spines--regions where protein sorting and synaptic delivery are known to occur. The specific localization of mLin-10 in Golgi is disrupted by a point mutation in an mLin-10 PDZ domain, indicating that a PDZ domain mediates this localization. Interactions between mLin-10 and glutamate receptors in both intracellular and synaptic membrane fractions were detected through biochemical assays. GST-pull down and co-immunoprecipitation experiments in heterologous cells delineated the protein domains required for interaction. These results demonstrated that glutamate receptors interact directly with mLin-10 through a PDZ domain-mediated mechanism. A PDZ point mutation enhances surface delivery of exogenous glutamate receptors in transfected neurons, suggesting that mLin-10 may regulate AMPA receptor trafficking in vivo.

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies show that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. In the current work, we show that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. We also found that a second ion channel, TRPL, bound to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, we found that INAD formed homopolymers and the homomultimerization occurred through two PDZ domains. Thus, we propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein-coupled cascade is tethered.

PDZ domains are binding modules mostly involved in cell signaling and cell-cell junctions. These domains are able to recognize a wide variety of natural targets and, among the PDZ partners, viruses have been discovered to interact with their host via a PDZ domain. With such an array of relevant and diverse interactions, PDZ binding specificity has been thoroughly studied and a traditional classification has grouped PDZ domains in three major specificity classes. In this work, we have selected four human PDZ domains covering the three canonical specificity-class binding mode and a set of their corresponding binders, including host/natural, viral and designed PDZ motifs. Through calorimetric techniques, we have covered the entire cross interactions between the selected PDZ domains and partners. The results indicate a rather basic specificity in each PDZ domain, with two of the domains that bind their cognate and some non-cognate ligands and the two other domains that basically bind their cognate partners. On the other hand, the host partners mostly bind their corresponding PDZ domain and, interestingly, the viral ligands are able to bind most of the studied PDZ domains, even those not previously described. Some viruses may have evolved to use of the ability of the PDZ fold to bind multiple targets, with resulting affinities for the virus-host interactions that are, in some cases, higher than for host-host interactions.

PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs), important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited.