Structure function relations in PDZ-domain-containing proteins: Implications for protein networks in cellular signalling

Protein scaffolds as essential backbones for organization of supramolecular signalling complexes are a recurrent theme in several model systems. Scaffold proteins preferentially employ linear peptide binding motifs for recruiting their interaction partners. PDZ domains are one of the more commonly encountered peptide binding domains in several proteins including those involved in scaffolding functions. This domain is known for its promiscuity both in terms of ligand selection, mode of interaction with its ligands as well as its association with other protein interaction domains. PDZ domains are subject to several means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to the effect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical and biophysical basis for this selectivity as well as promiscuity has been investigated and reviewed extensively. The present review focuses on the plasticity inherent in PDZ domains and its implications for modular organization as well as evolution of cellular signalling pathways in higher eukaryotes.     

Energetic determinants of internal motif recognition by PDZ domains

PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.     

Selectivity and promiscuity in the interaction network mediated by protein recognition modules

A substantial fraction of protein interactions in the cell is mediated by families of protein modules binding to relatively short linear peptides. Many of these interactions have a high dissociation constant and are therefore suitable for supporting the formation of dynamic complexes that are assembled and disassembled during signal transduction. Extensive work in the past decade has shown that, although member domains within a family have some degree of intrinsic peptide recognition specificity, the derived interaction networks display substantial promiscuity. We review here recent advances in the methods for deriving the portion of the protein network mediated by these domain families and discuss how specific biological outputs could emerge in vivo despite the observed promiscuity in peptide recognition in vitro.     

Convergent and divergent ligand specificity among PDZ domains of the LAP and zonula occludens (ZO) families

We present a detailed comparative analysis of the PDZ domains of the human LAP proteins Erbin, Densin-180, and Scribble and the MAGUK ZO-1. Phage-displayed peptide libraries and in vitro affinity assays were used to define ligand binding profiles for each domain. The analysis reveals the importance of interactions with all four C-terminal residues of the ligand, which constitute a core recognition motif, and also the role of interactions with more upstream ligand residues that support and modulate the core binding interaction. In particular, the results highlight the importance of site(-1), which interacts with the penultimate residue of ligand C termini. Site(-1) was found to be monospecific in the Erbin PDZ domain (accepts tryptophan only), bispecific in the first PDZ domain of ZO-1 (accepts tryptophan or tyrosine), and promiscuous in the Scribble PDZ domains. Furthermore, it appears that the level of promiscuity within site(-1) greatly influences the range of potential biological partners and functions that can be associated with each protein. These findings show that subtle changes in binding specificity can significantly alter the range of biological partners for PDZ domains, and the insights enhance our understanding of this diverse family of peptide-binding modules.     

Plasticity of PDZ domains in ligand recognition and signaling

The PDZ domain is a protein-protein interacting module that plays an important role in the organization of signaling complexes. The recognition of short intrinsically disordered C-terminal peptide motifs is the archetypical PDZ function, but the functional repertoire of this versatile module also includes recognition of internal peptide sequences, dimerization and phospholipid binding. The PDZ function can be tuned by various means such as allosteric effects, changes of physiological buffer conditions and phosphorylation of PDZ domains and/or ligands, which poses PDZ domains as dynamic regulators of cell signaling. This review is focused on the plasticity of the PDZ interactions.     

Analysis of PDZ domain-ligand interactions using carboxyl-terminal phage display

PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.     

A specificity map for the PDZ domain family

PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.     

Characterization of PDZ domain‐peptide interaction interface based on energetic patterns

PDZ domain is one of the abundant modular domains that recognize short peptide sequences to mediate protein–protein interactions. To decipher the binding specificity of PDZ domain, we analyzed the interactions between 11 mouse PDZ domains and 217 peptides using a method called MIECSVM, which energetically characterizes the domain‐peptide interaction using molecular interaction energy components (MIECs) and predicts binding specificity using support vector machine (SVM). Cross‐validation and leave‐one‐domain‐out test showed that the MIEC‐SVM using all 44 PDZ‐peptide residue pairs at the interaction interface outperformed the sequence‐based methods in the literature. A further feature (residue pair) selection procedure illustrated that 16 residue pairs were uninformative to the binding specificity, even though they contributed significantly (～50%) to the binding energy. If only using the 28 informative residue pairs, the performance of the MIEC‐SVM on predicting the PDZ binding specificity was significantly improved. This analysis suggests that the informative and uninformative residue interactions between the PDZ domain and the peptide may represent those contributing to binding specificity and affinity, respectively. We performed additional structural and energetic analyses to shed light on understanding how the PDZ‐peptide recognition is established. The success of the MIEC‐SVM method on PDZ domains in this study and SH3 domains in our previous studies illustrates its generality on characterizing protein‐ peptide interactions and understanding protein recognition from a structural and energetic viewpoint.     

Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.     

iSPOT: A web tool to infer the interaction specificity of families of protein modules

iSPOT (http://cbm.bio.uniroma2.it/ispot) is a web tool developed to infer the recognition specificity of protein module families; it is based on the SPOT procedure that utilizes information from position-specific contacts, derived from the available domain/ligand complexes of known structure, and experimental interaction data to build a database of residue-residue contact frequencies. iSPOT is available to infer the interaction specificity of PDZ, SH3 and WW domains. For each family of protein domains, iSPOT evaluates the probability of interaction between a query domain of the specified families and an input protein/peptide sequence and makes it possible to search for potential binding partners of a given domain within the SWISS-PROT database. The experimentally derived interaction data utilized to build the PDZ, SH3 and WW databases of residue-residue contact frequencies are also accessible. Here we describe the application to the WW family of protein modules.     

An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for understanding the interaction networks they establish. We determined the binding preference of the five PDZ domains in the protein tyrosine phosphatase PTP-BL by screening a random C-terminal peptide lambda phage display library. Interestingly, the potential of PDZ2 to interact with class III-type ligands was found to be modulated by the presence of PDZ1. Structural studies revealed a direct and specific interaction of PDZ1 with a surface on PDZ2 that is opposite the peptide binding groove. Long-range allosteric effects that cause structural changes in the PDZ2 peptide binding groove thus explain the altered PDZ2 binding preference. Our results experimentally corroborate that the molecular embedding of PDZ domains is an important determinant of their ligand binding specificity.     

Role of electrostatic interactions in PDZ domain ligand recognition

PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse alpha1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements.     

Sequence-specific long range networks in PSD-95/discs large/ZO-1 (PDZ) domains tune their binding selectivity

Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.     

Characterization of PDZ domain-peptide interaction interface based on energetic patterns

PDZ domain is one of the abundant modular domains that recognize short peptide sequences to mediate protein-protein interactions. To decipher the binding specificity of PDZ domain, we analyzed the interactions between 11 mouse PDZ domains and 2387 peptides using a method called MIEC-SVM, which energetically characterizes the domain-peptide interaction using molecular interaction energy components (MIECs) and predicts binding specificity using support vector machine (SVM). Cross-validation and leave-one-domain-out test showed that the MIEC-SVM using all 44 PDZ-peptide residue pairs at the interaction interface outperformed the sequence-based methods in the literature. A further feature (residue pair) selection procedure illustrated that 16 residue pairs were uninformative to the binding specificity, even though they contributed significantly (~50%) to the binding energy. If only using the 28 informative residue pairs, the performance of the MIEC-SVM on predicting the PDZ binding specificity was significantly improved. This analysis suggests that the informative and uninformative residue interactions between the PDZ domain and the peptide may represent those contributing to binding specificity and affinity, respectively. We performed additional structural and energetic analyses to shed light on understanding how the PDZ-peptide recognition is established. The success of the MIEC-SVM method on PDZ domains in this study and SH3 domains in our previous studies illustrates its generality on characterizing protein-peptide interactions and understanding protein recognition from a structural and energetic viewpoint.     

Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptides

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P(0) and P(-2) position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P(-1) and P(-4) position and a valine residue at the P(0) position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex.     

A Structural Portrait of the PDZ Domain Family

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.     

Distinct binding specificity of the multiple PDZ domains of INADL, a human protein with homology to INAD from Drosophila melanogaster

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda. We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.     

Structural Basis for Exquisite Specificity of Affinity Clamps, Synthetic Binding Proteins Generated through Directed Domain-interface Evolution

We have established a new protein-engineering strategy termed “directed domain-interface evolution” that generates a binding site by linking two protein domains and then optimizing the interface between them. Using this strategy, we have generated synthetic two-domain “affinity clamps” using PDZ and fibronectin type III (FN3) domains as the building blocks. While these affinity clamps all had significantly higher affinity toward a target peptide than the underlying PDZ domain, two distinct types of affinity clamps were found in terms of target specificity. One type conserved the specificity of the parent PDZ domain, and the other increased the specificity dramatically. Here, we characterized their specificity profiles using peptide phage-display libraries and scanning mutagenesis, which suggested a significantly enlarged recognition site of the high-specificity affinity clamps. The crystal structure of a high-specificity affinity clamp showed extensive contacts with a portion of the peptide ligand that is not recognized by the parent PDZ domain, thus rationalizing the improvement of the specificity of the affinity clamp. A comparison with another affinity clamp structure showed that, although both had extensive contacts between PDZ and FN3 domains, they exhibited a large offset in the relative position of the two domains. Our results indicate that linked domains could rapidly fuse and evolve as a single functional module, and that the inherent plasticity of domain interfaces allows for the generation of diverse active-site topography. These attributes of directed domain-interface evolution provide facile means to generate synthetic proteins with a broad range of functions.     

A PDZ domain-based assay for measuring HIV protease activity: assay design considerations

We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain*peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu(3+)chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ). The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain*peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.     

The multiple‐specificity landscape of modular peptide recognition domains

Modular protein interaction domains form the building blocks of eukaryotic signaling pathways. Many of them, known as peptide recognition domains, mediate protein interactions by recognizing short, linear amino acid stretches on the surface of their cognate partners with high specificity. Residues in these stretches are usually assumed to contribute independently to binding, which has led to a simplified understanding of protein interactions. Conversely, we observe in large binding peptide data sets that different residue positions display highly significant correlations for many domains in three distinct families (PDZ, SH3 and WW). These correlation patterns reveal a widespread occurrence of multiple binding specificities and give novel structural insights into protein interactions. For example, we predict a new binding mode of PDZ domains and structurally rationalize it for DLG1 PDZ1. We show that multiple specificity more accurately predicts protein interactions and experimentally validate some of the predictions for the human proteins DLG1 and SCRIB. Overall, our results reveal a rich specificity landscape in peptide recognition domains, suggesting new ways of encoding specificity in protein interaction networks.