Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1‐2 Supramodule of PSD‐95

PSD‐95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein–protein interactions in the brain with its PDZ domains. It has been suggested that PSD‐95 is composed of two supramodules, one of which is the PDZ1‐2 tandem domain. Here we have developed rigidified high‐affinity dimeric ligands that target the PDZ1‐2 supramodule, and established the biophysical parameters of the dynamic PDZ1‐2/ligand interactions. By employing ITC, protein NMR, and stopped‐flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD‐95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.     

Structural Diversity of PDZ–Lipid Interactions

PDZ domains are globular protein modules that are over‐and‐above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ–phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin‐1, syntenin‐2, PTP‐Bas, PAR‐3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ–lipid interactions and the consequences for peptide interactions.     

Small‐Molecule Inhibitors of AF6 PDZ‐Mediated Protein–Protein Interactions

PDZ (PSD‐95, Dlg, ZO‐1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ‐mediated protein–protein interactions has important implications in disease‐related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1‐fused gene from chromosome 6), which is an essential component of cell–cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile‐Gln‐Ser‐Val‐Glu‐Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6–Bcr interaction and interfere with epidermal growth factor (EGF)‐dependent signaling.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Directed evolution of PDZ variants to generate high-affinity detection reagents

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.     

Organization of signaling complexes by PDZ-domain scaffold proteins

Transduction of biological signals from receptors at the plasma membrane to their targets in cytoplasm and nucleus relies on specific protein-protein interactions. A common strategy used by cells is to organize proteins in the same signaling cascade into large molecular weight, multiprotein complexes. PDZ domain proteins have been shown to play important roles in assembling various signaling complexes. Here, we first present biophysical basis of the advantages of organizing proteins in a signaling cascade into a clustered multiprotein complex. We then discuss the structure, ligand binding, and function of PDZ domains in organizing synaptic signaling complexes.     

Interaction of Classical Platinum Agents with the Monomeric and Dimeric Atox1 Proteins: A Molecular Dynamics Simulation Study

We carried out molecular dynamics simulations and free energy calculations for a series of binary and ternary models of the cisplatin, transplatin and oxaliplatin agents binding to a monomeric Atox1 protein and a dimeric Atox1 protein to investigate their interaction mechanisms. All three platinum agents could respectively combine with the monomeric Atox1 protein and the dimeric Atox1 protein to form a stable binary and ternary complex due to the covalent interaction of the platinum center with the Atox1 protein. The results suggested that the extra interaction from the oxaliplatin ligand–Atox1 protein interface increases its affinity only for the OxaliPt + Atox1 model. The binding of the oxaliplatin agent to the Atox1 protein might cause larger deformation of the protein than those of the cisplatin and transplatin agents due to the larger size of the oxaliplatin ligand. However, the extra interactions to facilitate the stabilities of the ternary CisPt + 2Atox1 and OxaliPt + 2Atox1 models come from the α1 helices and α2-β4 loops of the Atox1 protein–Atox1 protein interface due to the cis conformation of the platinum agents. The combinations of two Atox1 proteins in an asymmetric way in the three ternary models were analyzed. These investigations might provide detailed information for understanding the interaction mechanism of the platinum agents binding to the Atox1 protein in the cytoplasm.     

Computer modelling of the receptor-binding domains of VEGF and PIGF

Models of the platelet-derived growth factor (PDGF)-like domains of vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) were built based on their homology to PDGF. These domains contain most of the determinants for receptor binding. The sequences of these proteins exhibit limited but significant homology to that of platelet-derived growth factor (PDGF), a member of the cystine knot growth factor family. The eight cysteine residues that are involved in intra- and interchain disulphide bonds are conserved. Two high affinity receptors for VEGF have been identified, only one of which binds PIGF. The models presented here are consistent with results that show that VEGF receptor binding is mediated by charged residues in the loops. A comparison of the models suggests that the difference in receptor- binding specificity between VEGF and PIGF may be due to differences in the distribution of positively charged residues and the exposure of hydrophobic residues in the loops.      

Phosphorylation of the WWOX Protein Regulates Its Interaction with p73

We describe a molecular characterization of the interaction between the cancer-related proteins WWOX and p73. This interaction is mediated by the first of two WW domains (WW1) of WWOX and a PPXY-motif-containing region in p73. While phosphorylation of Tyr33 of WWOX and association with p73 are known to affect apoptotic activity, the quantitative effect of phosphorylation on this specific interaction is determined here for the first time. Using ITC and fluorescence anisotropy, we measured the binding affinity between WWOX domains and a p73 derived peptide, and showed that this interaction is regulated by Tyr phosphorylation of WW1. Chemical synthesis of the phosphorylated domains of WWOX revealed that the binding affinity of WWOX to p73 is decreased when WWOX is phosphorylated. This result suggests a fine-tuning of binding affinity in a differential, ligand-specific manner: the decrease in binding affinity of WWOX to p73 can free both partners to form new interactions.     

Deep Sequencing of a Systematic Peptide Library Reveals Conformationally-Constrained Protein Interface Peptides that Disrupt a Protein-Protein Interaction

Disrupting protein-protein interactions is difficult due to the large and flat interaction surfaces of the binding partners. The BLIP and BLIP-II proteins are unrelated in sequence and structure and yet each potently inhibit β-lactamases. High-throughput oligonucleotide synthesis was used to construct a 12,470-member library containing overlapping linear and cyclic peptides ranging in size from 6 to 21 amino acids that scan through the sequences of BLIP and BLIP-II. Phage display affinity selections and deep sequencing revealed that, despite the differences in interaction surfaces with β-lactamases, rapid enrichment of consensus peptide regions originating from both BLIP and BLIP-II contact residues in the binding interface occurred. BLIP and BLIP-II peptides that were enriched by affinity selection were shown to bind β-lactamases and disrupt the BLIP/β-lactamase interaction. The results suggest that peptides that bind at and disrupt PPI interfaces can be identified through systematic peptide library construction, affinity selection, and deep sequencing.     

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV‐1 Integrase

The p75 splice variant of lens epithelium‐derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre‐integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X‐ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV‐1 integrase core domain. Although the X‐ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low‐energy solution and bound conformations.     

Characterization of a putative phosphorylation switch: adaptation of SPOT synthesis to analyze PDZ domain regulation mechanisms

Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g., PDZ, SH2, SH3, WW), which are often regulated (positively or negatively) by phosphorylation. To address the in vitro analysis of PDZ domain regulation by such phosphorylation, we improved the inverted peptide method. This method is based on standard SPOT synthesis, followed by inversion of the peptide under acidic conditions to generate the free C termini necessary for PDZ domain ligand recognition. The benefit of the newly introduced acidic conditions is the preservation of the incorporated phosphate group during peptide synthesis. Furthermore, the improved method is more robust and shows an increased signal-to-noise ratio. As representative examples, we used the AF6, ERBIN, and SNA1 (alpha-1-syntrophin) PDZ domains to analyze the influence of ligand-position-dependent phosphorylation. We could clearly demonstrate severe down-regulation by phosphorylation of the PDZ ligand position -2 (<50 %) and slightly less at position -1 ( approximately 50 %). These results are specific and reproducible for all three PDZ domains. Finally, we confirmed the influence of negative regulation by using the protein kinase BCR as the AF6 PDZ domain ligand. For the first time, this approach allows the SPOT synthesis technique to be used to screen large libraries of phosphorylated peptides in vitro. This should ultimately help in the identification of phosphorylation-dependent regulation mechanisms in vivo.     

Glycosylation assists binding of HIV protein gp120 to human CD4 receptor

The role of glycosylation of proteins on its binding affinity is not well understood. Even a monosaccharide (magenta) placed at a glycosylation site can significantly enhance binding of peptides to their receptor. If glycosylated, an HIV protein binds stronger and faster to its primary receptors on human cells.     

Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding

The impact of the incorporation of a non‐natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post‐translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X‐ray crystallography. The structure and dynamics of the apo state of AZH‐modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH‐modified PDZ3 and an azulene‐linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene‐linked peptide and the isotopically labelled protein. Co‐crystallisation and soaking failed for the peptide‐bound holo complex. NMR spectroscopy, however, allowed determination of the protein–ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA‐modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target.     

A Peptide-PNA Hybrid Beacon for Sensitive Detection of Protein Biomarkers in Biological Fluids

Specific and rapid detection of proteins in biological fluids poses a challenging problem. In biological fluids, many proteins are present at low concentrations, requiring high affinity and specificity of the beacon-protein interaction. We report the design of a peptide-PNA hybrid beacon that exploits the dimeric nature of a target protein, S100B, a biomarker for brain trauma, to enhance binding affinity and specificity. The complementary base-pairing of the PNA bases brings the two arms of the beacon, one carrying an Alexa tag and the other carrying a Dabcyl moiety, into proximity, thus quenching Alexa fluorescence. Each of the arms carries a sequence that binds to one of the subunits. Binding to the target separates the quencher from the probe lifting the quenching of fluorescence. Enhanced affinity and specificity resulting from simultaneously binding to two sites allowed specific detection of S100B at low-nanomolar concentrations in the presence of serum. The design can be easily adapted for the detection of proteins containing multiple binding sites and could prove useful for rapid and sensitive biomarker detection.     

Discovery, structure-activity relationship studies, and crystal structure of nonpeptide inhibitors bound to the Shank3 PDZ domain

Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C‐terminus of guanylate‐kinase‐associated protein (GKAP). Here, small‐molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high‐affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein–protein interaction. Chemical synthesis via a hetero‐Diels–Alder strategy is employed for hit optimization, and structure–activity relationship studies are performed. Best hits possess K_i values in the 10 μM range, and binding to the PDZ domain is confirmed by ¹H,¹⁵N HSQC NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD‐95, syntrophin, and DVL3 was studied using ¹H,¹⁵N HSQC NMR spectroscopy.     

Chemical biology approaches reveal conserved features of a C-terminal processing PDZ protease

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C-terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram-negative bacteria. While several HtrA proteases have been extensively characterized, many features of C-terminal processing proteases such as tail-specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid-based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed.     

Human Serine Racemase Weakly Binds the Third PDZ Domain of PSD-95

Human serine racemase (hSR) is a pyridoxal-5′-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg²⁺, ATP, post-translational modifications, and the interaction with protein partners. In particular, the C-terminus of murine SR binds the third PDZ domain (PDZ3) of postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase (MAGUK) family involved in the trafficking and localization of glutamate receptors. The structural details of the interaction and the stability of the complex have not been elucidated yet. We evaluated the binding of recombinant human PSD-95 PDZ3 to hSR by glutaraldehyde cross-linking, pull-down assays, isothermal titration calorimetry, nuclear magnetic resonance, and enzymatic assays. Overall, a weak interaction was observed, confirming the binding for the human orthologs but supporting the hypothesis that a third protein partner (i.e., stargazin) is required for the regulation of hSR activity by PSD-95 and to stabilize their interaction.     

Aminoglycoside-quinacridine conjugates: towards recognition of the P6.1 element of telomerase RNA

A modular synthesis has been developed which allows easy and rapid attachment of one or two aminoglycoside units to a quinacridine intercalator, thereby leading to monomeric and dimeric conjugates. Melting temperature (Tm) experiments show that the tobramycin dimeric conjugate TD1 exhibits strong binding to the P6.1 element of human telomerase RNA. By contrast, tobramycin alone is much less efficient and the monomeric compound TM1 elicits a poor binding ability. Monitoring of the interaction by an electrophoretic mobility shift assay shows a 1:1 stoichiometry for the binding of the dimeric compound to the hairpin structure and confirms the lower affinity for a control duplex. Protection experiments with RNase T1 indicate interaction of the drug both in the stem and in the loop of the hairpin. Taken together, the data suggest a binding of TD1 inside the hairpin at the stem-loop junction. The same trends are observed with paromomycin and kanamycin analogues but with a lower affinity.