Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.     

Design and structure of peptide and peptidomimetic antagonists of protein-protein interaction

Peptides based on the amino acid sequences found at protein-protein interaction sites make excellent leads for antagonist development. A statistical picture of amino acids involved in protein-protein interactions indicates that proteins recognize and interact with one another through the restricted set of specialized interface amino acid residues, Pro, Ile, Tyr, Trp, Asp and Arg. These amino acids represent residues from each of the three classes of amino acids, hydrophobic, aromatic and charged, with one anionic and one cationic residue at neutral pH. The use of peptides as drug leads has been successfully used to search for antagonists of cell-surface receptors. Peptide, peptidomimetic, and non-peptide organic inhibitors of a class of cell surface receptors, the integrins, currently serve as therapeutic and diagnostic imaging agents. In this review, we discuss the structural features of protein-protein interactions as well as the design of peptides, peptidomimetics, and small organic molecules for the inhibition of protein-protein interactions. Information gained from studying inhibitors of integrin functions is now being applied to the design and testing of inhibitors of other protein-protein interactions. Most drug development progress in the past several decades has been made using the enzyme binding-pocket model of drug targets. Small molecules are designed to fit into the substrate-binding pockets of proteins based on a lock-and-key, induced-fit, or conformational ensemble model of the protein binding site. Traditionally, enzymes have been used as therapeutic drug targets because it was easier to develop rapid, sensitive screening assays, and to find low molecular weight inhibitors that blocked the active site. However, for proteins which interact with other proteins, rather than with small substrate molecules, the lack of binding pockets means that this approach will not generally succeed. There exist many diseases in which the inhibition of protein-protein interactions would provide therapeutic benefit, but there are no general methods available to address such problems. The focus of the first part of this review is to discuss the features of protein-protein interactions which may serve as general guidelines for the development and design of inhibitors for protein-protein interactions. In the second part we focus on the design of peptides (lead compounds) and their conversion into peptidomimetics or small organic molecules for the inhibition of protein-protein interactions. We draw examples from the important and emerging area of integrin-based cell adhesion and show how the principles of protein-protein interactions are followed in the discovery, optimization and usage of specific protein interface peptides as drug leads.     

A systematic family-wide investigation reveals that ~30% of mammalian PDZ domains engage in PDZ-PDZ interactions

PDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C termini of their target proteins. However, in a few instances, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical-binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify interactions and coaffinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (~30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand.     

An efficient approach to chiral C8/C9-piperazino-substituted 1,4-benzodiazepin-2-ones as peptidomimetic scaffolds

A promising way to interfere with biological processes is through the modulation of protein-protein interactions by means of small molecules acting as peptidomimetics. The 1,4-benzodiazepine scaffold has been widely reported as a peptide-mimicking, pharmacogenic system. While several synthetic pathways to C6-8 substituted benzodiazepines have been disclosed, few 1,4-benzodiazepines substituted at C9 have been reported. Herein, we describe a versatile approach to introduce cyclic, protonatable functionality at C8/C9. Introduction of the piperazine system at C8 and C9 gave access to a unique functionalization of the versatile benzodiazepine skeleton, broadening tailoring options on the benzofused side of the molecule, and the possibility of discovering novel peptidomimetics potentially able to modulate protein-protein interactions. Coupling of activated amino acids with poorly reactive anilines under mild conditions, while avoiding racemization, gave easy access to these compounds. Efficient amino acid activation was obtained by exploiting the rapid formation of acid chlorides under low temperature and acid/base free conditions, using triphenylphosphine and hexachloroacetone. This procedure successfully resulted in high reaction yields, did not produce racemization (ee > 98%, as demonstrated by using chiral solvating agents), and was compatible with the acid sensitive protecting groups present in the substrates.     

A selective irreversible inhibitor targeting a PDZ protein interaction domain

Irreversible inhibitors of proteases have proven themselves useful tools for determining which proteases are active under given conditions in tissues or cells and for studying the functional role that a protease plays in physiological processes. The application of such techniques to the study of the activity and function of protein-protein interactions has been hindered by the lack of guiding principles for the mechanistic design of irreversible inhibitors targeting the "active site" of a protein interaction. We report herein the first example of a mechanism-based irreversible inhibitor of a protein interaction that has been specifically targeted to one member of the PDZ family of protein interaction domains: the second PDZ domain of the membrane-associated guanylate kinase MAGI3. This inhibitor was designed using rationally directed computational evaluation to take advantage of a conserved histidine in the PDZ domain by introducing an ionizable group that will be held in close proximity to that nucleophile during binding. The novel compound exhibits all of the characteristics of an irreversible inhibitor of the interaction of the tumor suppressor PTEN with MAGI3 in in vitro models. In cells, the inhibitor can be shown to release PTEN from sequestration by MAGI3 and consequently upregulate the PKB signaling pathway.     

Bridged peptide macrocycles as ligands for PDZ domain proteins

[reaction: see text] Conformationally constrained side chain-bridged cyclic peptides were prepared using bis-carboxylic acid ring spacers. These macrocyles were designed to inhibit protein-protein interactions mediated by the third PDZ domain (PDZ3) of a mammalian neuronal protein, PSD-95. Isothermal titration calorimetry (ITC) experiments measured dissociation constants in the low micromolar range. For each compound, the change in entropy (TdeltaS) of binding either is comparable in magnitude to the enthalpy change (deltaH) or is the predominant driving force for association.     

Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays

One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.     

An improved method for the synthesis of cellulose membrane-bound peptides with free C termini is useful for PDZ domain binding studies

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.     

Predicting protein-protein interactions using graph invariants and a neural network

The PDZ domain of proteins mediates a protein-protein interaction by recognizing the hydrophobic C-terminal tail of the target protein. One of the challenges put forth by the DREAM (Discussions on Reverse Engineering Assessment and Methods) 2009 Challenge consists of predicting a position weight matrix (PWM) that describes the specificity profile of five PDZ domains to their target peptides. We consider the primary structures of each of the five PDZ domains as a numerical sequence derived from graph-theoretic models of each of the individual amino acids in the protein sequence. Using available PDZ domain databases to obtain known targets, the graph-theoretic based numerical sequences are then used to train a neural network to recognize their targets. Given the challenge sequences, the target probabilities are computed and a corresponding position weight matrix is derived. In this work we present our method. The results of our method placed second in the DREAM 2009 challenge.     

Syntheses and activities of new C10 beta-turn peptidomimetics

A program to identify small molecules that mimic or disrupt protein-protein interactions led us to design the peptidomimetics 1-3. Solid-phase syntheses of 1-3 were developed. The purities of the crude materials isolated from the resin tend to be highest for the S- and N-compounds 2 and 3 and better than in the corresponding syntheses of peptidomimetics A. The particular dipeptide units incorporated were chosen to correspond with the turn regions of the neurotrophins (e.g., nerve growth factor [NGF] and the neurotrophin factor-3 [NT-3]). Preliminary studies were performed to access the binding of these analogues to Trk receptors and their ability to induce cell survival (just as NGF and NT-3 do). Several active compounds were identified. However, poor water solubilities of some of the other compounds preclude reliable testing. Consequently, solid-phase modifications to the synthetic procedures were investigated to provide access to the derivatives 12-14 in which the aromatic nitro group is replaced by amine, guanidine, or sulfonamide functionalities. The latter are more acceptable pharmacophores than nitro groups and also tend to increase the water solubilities of the peptidomimetics.     

Inhibitors of tyrosine kinase proteins induced Ras signaling pathway as potential anti-tumor agents

Cellular signaling pathways induced by growth-factor receptors with tyrosine kinase activity are frequently deregulated in cancer. Anti-tumor agents that inhibit their enzymatic tyrosine kinase activity have been designed and are now used in human chemotherapy. We review here our data constituting an alternative way to interrupt over-expressed signaling pathway by inhibiting protein-protein interactions. In our approach, the adaptor protein Grb2 over-expressed in connection with HER2/ErbB2/neu in Ras signaling pathway was chosen as a target. Peptides and peptidomimetics with very high affinities for either SH3 or SH2 domains of Grb2 were rationally designed from structural data. We describe their synthesis, their capacity to interrupt the signaling pathway and their anti-proliferative activity. To cite this article: M. Vidal et al., C. R. Chimie 8 (2005).     

Simultaneous prediction of binding free energy and specificity for PDZ domain–peptide interactions

Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain–peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with Rosetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several Rosetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain–peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain–peptide interactions.     

A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors

Background: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets.Results: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors.Conclusions: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.     

A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain

PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters (Z'≈0.7 and Z≈0.6). Out of ~200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ~5.0 μM to ~193 μM. Binding to the PICK1 PDZ domain was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 μM) in the FP assay, and was according to fluorescence resonance energy transfer (FRET) experiments capable of inhibiting the interaction between the C-terminus of the GluR2 subunit of the AMPA-type glutamate receptor and PICK1 in live cells. Additional experiments suggested that CSC-03 most likely is an irreversible inhibitor but with specificity for PICK1 since it did not bind three different PDZ domains of PSD-95. Summarized, our data suggest that FP based screening assays might be a widely applicable tool in the search for small molecule inhibitors of PDZ domain interactions.     

Synthesis of amino acid-derived cyclic acyl amidines for use in beta-strand peptidomimetics

The acyl amidine represented by the 4,5-dihydro-2(3H)-pyrazinone ring system 2 is isosteric to the vinylogous amide of the 1,2-dihydro-3(6H)-pyridinone 1, but its assembly from separate amine and amide components enables ready incorporation of an amino acid side chain with correct regio- and stereochemistry. beta-Strand peptidomimetics incorporating amino acid analogues based on 2 have recently been shown to be potent, protease-resistant ligands to a PDZ protein-interaction domain. Two routes to the protected dipeptide analogue 3 are described.     

A Modular Synthesis of Conformationally Preorganised Extended β‐Strand Peptidomimetics

A promising strategy for mediating protein–protein interactions is the use of non‐peptidic mimics of secondary structural protein elements, such as the α‐helix. Recent work has expanded the scope of this approach by providing proof‐of‐principle scaffolds that are conformationally biased to mimic the projection of side‐chains from one face of another common secondary structural element—the β‐strand. Herein, we present a synthetic route that has key advantages over previous work: monomers bearing an amino acid side‐chain were pre‐formed before rapid assembly to peptidomimetics through a modular, iterative strategy. The resultant oligomers of alternating pyridyl and six‐membered cyclic ureas accurately reproduce a recognition domain of several amino acid residues of a β‐strand, demonstrated herein by mimicry of the i, i+2, i+4 and i+6 residues.     

Synthetic non-peptide mimetics of alpha-helices

Proteins in nature fold into native conformations in which combinations of peripherally projected aliphatic, aromatic and ionic functionalities direct a wide range of properties. Alpha-helices, one of the most common protein secondary structures, serve as important recognition regions on protein surfaces for numerous protein-protein, protein-DNA and protein-RNA interactions. These interactions are characterized by conserved structural features within the alpha-helical domain. Rational design of structural mimetics of these domains with synthetic small molecules has proven an effective means to modulate such protein functions. In this tutorial review we discuss strategies that utilize synthetic small-molecule antagonists to selectively target essential protein-protein interactions involved in certain diseases. We also evaluate some of the protein-protein interactions that have been or are potential targets for alpha-helix mimetics.     

Stable hairpins with beta-peptides: route to tackle protein-protein interactions

Experimental and theoretical data demonstrate that sequences of heterochiral beta (2,3)-amino acids and a turn-inducing beta-dipeptide adopt hairpin-like structures in methanol. On the basis of extensive canonical and replica exchange MD simulations, we could transfer these findings to water as the solvent of physiological relevance. We show that rationally designed beta-peptides exhibit a higher folding tendency and a more robust hairpin structure formation in water compared with alpha-peptides. Furthermore, our designed scaffold enables the addition of a wide variety of functions without disrupting the structure. Since hairpins are often involved in protein interactions, the very stable hairpin-like fold of our designed beta-peptides might be used as a lead scaffold for the design of molecules that specifically modulate protein-protein interactions. This is demonstrated by application of this concept to the recognition of proline-rich sequences (PRS) by WW domains, an important interaction in cell signaling. We focus on the possibility to imitate the strands 2 and 3 of any WW domain as a minimal motif to recognize their target sequences PPXY. We conclude that rationally designed beta-peptide hairpins can serve as scaffolds not only to tackle PPII recognition but also to open up a way to influence a wide variety of protein-protein interactions.     

Structure-based design of conformationally constrained cyclic peptidomimetics to target the MLL1-WDR5 protein–protein interaction as inhibitors of the MLL1 methyltransferase activity

We described herein structure-based design,synthesis and evaluation of conformationally constrained,cyclic peptidomimetics to block the MLL1-WDR5 protein–protein interaction as inhibitors of the MLL1 histone methyltransferase activity.Our study has yielded cyclic peptidomimetics with very high binding affinities to WDR5(Kivalues 1 nmol/L) and function as antagonists of the MLL1 histone methyltransferase activity.