Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.     

An In Silico Analysis of the Binding Modes and Binding Affinities of Small Molecule Modulators of PDZ-Peptide Interactions

Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson’s disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K_i or K_d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K_i or K_d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.     

Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95

PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.     

Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands

PDZ domains are modular protein-protein interaction domains that bind to specific C-terminal sequences of membrane proteins and/or to other PDZ domains. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal peptide ligands and heterodimerize with the extended nNOS PDZ domain. The capacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate- binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K(+) channel Kv1.4. The same mutation affected nNOS binding to alpha1- and beta1-syntrophin PDZ domains to a lesser extent, due in part to the stabilizing effect of tertiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin that forms a tertiary contact with the nNOS PDZ domain. This residue is important for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basis, we have identified the C terminus of the Kir2.1 channel as a possible binding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.     

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.     

The kinetics of PDZ domain-ligand interactions and implications for the binding mechanism

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A + B --> A.B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex.     

Synthesis and evaluation of bidentate ligands designed to interact with PDZ domains

We designed bidentate ligands to target PDZ domains through two binding sites: site S0, delimited by the GLGF loop, and site S1, a zone situated around loop β(B)/β(C). A molecular docking study allowed us to design a generic S0 binder, to which was attached a variable size linker, itself linked to an amino acid aimed to interact with the S1 site of PDZ domains. A series of 15 novel bidentate ligands was prepared in 6-11 steps in good overall yield (24-43%). Some of these ligands showed an inhibitory activity against serotonin 5-HT2A receptor/PSD-95 interaction. This was assessed by pull-down assay using a synthetic decapeptide corresponding to the C-terminal residues of the receptor as a bait.     

Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.

Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding.     

NMR evaluation of interactions between substituted-indole and PDZ1 domain of PSD-95

We synthesized small organic molecules designed as PDZ ligands. These indole-based compounds were evaluated for their interaction with the PDZ1 domain of the post-synaptic density 95 (PSD-95) protein. Three molecules were found to interact with the targeted PDZ protein by NMR. One of them showed chemical shift perturbations closely related to the natural ligands.     

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.     

Rewiring of PDZ domain-ligand interaction network contributed to eukaryotic evolution

PDZ domain-mediated interactions have greatly expanded during metazoan evolution, becoming important for controlling signal flow via the assembly of multiple signaling components. The evolutionary history of PDZ domain-mediated interactions has never been explored at the molecular level. It is of great interest to understand how PDZ domain-ligand interactions emerged and how they become rewired during evolution. Here, we constructed the first human PDZ domain-ligand interaction network (PDZNet) together with binding motif sequences and interaction strengths of ligands. PDZNet includes 1,213 interactions between 97 human PDZ proteins and 591 ligands that connect most PDZ protein-mediated interactions (98%) in a large single network via shared ligands. We examined the rewiring of PDZ domain-ligand interactions throughout eukaryotic evolution by tracing changes in the C-terminal binding motif sequences of the PDZ ligands. We found that interaction rewiring by sequence mutation frequently occurred throughout evolution, largely contributing to the growth of PDZNet. The rewiring of PDZ domain-ligand interactions provided an effective means of functional innovations in nervous system development. Our findings provide empirical evidence for a network evolution model that highlights the rewiring of interactions as a mechanism for the development of new protein functions. PDZNet will be a valuable resource to further characterize the organization of the PDZ domain-mediated signaling proteome.     

Interdomain interactions in the tumor suppressor discs large regulate binding to the synaptic protein GukHolder

Multidomain scaffolding proteins are central components of many signaling pathways and are commonly found at membrane specializations. Here we have shown that multiple interdomain interactions in the scaffold Discs Large (Dlg) regulate binding to the synaptic protein GukHolder (GukH). GukH binds the Src homology 3 (SH3) and guanylate kinase-like (GK) protein interaction domains of Dlg, whereas an intramolecular interaction between the two domains inhibits association with GukH. Regulation occurs through a PDZ domain adjacent to the SH3 that allows GukH to interact with the composite SH3-GK binding site, but PDZ ligands inhibit GukH binding such that Dlg forms mutually exclusive PDZ ligand and GukH cellular complexes. The PDZ-SH3-GK module is a common feature of membrane associate guanylate kinase scaffolds such as Dlg, and these results indicate that its supramodular architecture leads to regulation of Dlg complexes.     

NHERF1/EBP50 head-to-tail intramolecular interaction masks association with PDZ domain ligands

Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na(+)/H(+) exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and beta-catenin. An EB region composite structure comprising an alpha-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.     

The interaction between PSD-95 and Ca2+/calmodulin is enhanced by PDZ-binding proteins

In this study, we evaluate the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin. Surface plasmon resonance spectroscopy was used to characterize the binding of PSD-95 to calmodulin that had been immobilized on a sensor chip. Additionally, soluble calmodulin was found to inhibit the binding of PSD-95 to immobilized calmodulin. The HOOK region of PSD-95, which is located between the src homology 3 domain and the guanylate kinase-like domain, was determined to be involved in the binding of PSD-95 to calmodulin. We also found that C-terminal peptides from proteins such as CRIPT and the N-methyl-d-aspartate receptor NR2B subunit, which associate with the PDZ domain of PSD-95, enhanced the affinity of PSD-95 for calmodulin. The binding of ligands to the PDZ domain may change the conformation of PSD-95 and affect the interaction between PSD-95 and calmodulin.     

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.     

Two conserved residues govern the salt and pH dependencies of the binding reaction of a PDZ domain

PDZ domains are protein-protein interaction modules found in hundreds of human proteins. Their binding reactions are sensitive to variations in salt and pH but the basis of the respective dependence has not been clear. We investigated the binding reaction between PSD-95 PDZ3 and a peptide corresponding to a native ligand with protein engineering in conjunction with stopped-flow and equilibrium fluorimetry and found that the two conserved residues Arg-318 and His-372 were responsible for the salt and pH dependencies, respectively. The basis of the salt-dependent variation of the affinity was explored by mutating all charged residues in and around the peptide-binding pocket. Arg-318 was found to be crucial, as mutation to alanine obliterated the effect of chloride on the binding constants. The direct interaction of chloride with Arg-318 was demonstrated by time-resolved urea denaturation experiments, where the Arg-318 --> Ala mutant was less stabilized by addition of chloride as compared with wild-type PDZ3. We also demonstrated that protonation of His-372 was responsible for the increase of the equilibrium dissociation constant at low pH. Both chloride concentration and pH (during ischemia) vary in the postsynaptic density, where PSD-95 is present, and the physiological buffer conditions may thus modulate the interaction between PSD-95 and its ligands through binding of chloride and protons to the "molecular switches" Arg-318 and His-372, respectively.     

Enhanced RNA binding of dimerized aminoglycosides.

Aminoglycoside antibiotics have recently emerged as an intriguing family of RNA binding molecules and they became leading structures for the design of novel RNA ligands. The demystification of the aminoglycoside-RNA recognition phenomenon is required for the development of superior binders. To explore the existence of multiple binding sites in a large RNA molecule, we have synthesized covalently linked symmetrical and nonsymmetrical dimeric aminoglycosides. These unnatural derivatives were compared to their natural "monomeric" counterparts in their ability to inhibit the Tetrahymena ribozyme. The dimeric aminoglycosides inhibit ribozyme function 20 to 1.2 x 10(3) fold more effectively than their natural parent compounds. The inhibition curves of dimeric aminoglycosides have characteristic shapes suggesting the presence of at least two high affinity-binding sites within the ribozyme's three-dimensional fold. The interaction of a dimeric aminoglycoside with two complementary sites of the RNA molecule is proposed. This binding motif may have implications on the development of new drugs targeting pivotal RNA molecules of bacterial and viral pathogens.     

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.     

The binding affinities of proteins interacting with the PDZ domain of PICK1

Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo-1 (PDZ) protein-protein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target.     

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

A substantial body of work has been devoted to the design and synthesis of glycosyltransferase inhibitors. A major obstacle has always been the demanding chemistry. Therefore, only few potent and selective inhibitors are known to date. Glycosyltransferases possess two distinct binding sites, one for the donor substrate, and one for the acceptor substrate. In many cases binding to the donor site is well defined but data for acceptor binding is sparse. In particular, acceptor binding sites are often shallow, and in many cases the dimensions of the binding pocket are not well defined. One approach to glycosyltransferase inhibitors is to chemically link donor site and acceptor site ligands to generate high affinity binders. Here, we describe a novel approach to identify acceptor site ligands from a fragment library. We have chosen human blood group B galactosyltransferase (GTB) as a biologically important model target. The approach utilizes a combination of STD NMR, spin-lock filtered NMR experiments and surface plasmon resonance measurements. Following this route we have identified molecular fragments from a fragment library that bind to the acceptor site of GTB with affinities of the order of a natural acceptor substrate. Unlike natural substrates these fragments allow for straightforward chemical modifications and, therefore will serve as scaffolds for potent GTB inhibitors. In general, the approach described is applicable to any glycosyltransferase and may assist in the development of novel glycosyltransferase inhibitors.