Phage display identification of high-affinity ligands for human TSG101-UEV: A structural and thermodynamic study of PTAP recognition.

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disrupting TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of novel host-oriented antivirals with a broad spectrum of action. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. Here we present a detailed thermodynamic, structural, and dynamic characterization viral PTAP Late domain recognition by TSG101-UEV, combining isothermal titration calorimetry, X-ray diffraction structural studies, molecular dynamics simulations, and computational analysis of intramolecular communication pathways. Our analysis highlights key contributions from conserved hydrophobic contacts and water-mediated hydrogen bonds at the PTAP binding interface. We have identified additional electrostatic hotspots adjacent to the core motif that modulate affinity. Using competitive phage display screening we have improved affinity by 1-2 orders of magnitude, producing novel peptides with low micromolar affinities that combine critical elements found in the best natural binders. Molecular dynamics simulations revealed that optimized peptides engage new pockets on the UEV domain surface. This study provides a comprehensive view of the molecular forces directing TSG101-UEV recognition of PTAP motifs, revealing that binding is governed by conserved structural elements yet tuneable through targeted optimization. These insights open new venues to design inhibitors targeting TSG101-dependent pathways with potential application as novel broad-spectrum antivirals.

Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

Understanding binding affinity and specificity of modular protein domains: A focus in ligand design for the polyproline-binding families.

Within the modular protein domains there are five families that recognize proline-rich sequences: SH3, WW, EVH1, GYF and UEV domains. This chapter reviews the main strategies developed for the design of ligands for these families, including peptides, peptidomimetics and drugs. We also describe some studies aimed to understand the molecular reasons responsible for the intrinsic affinity and specificity of these domains.

Phage display identification of nanomolar ligands for human NEDD4-WW3: Energetic and dynamic implications for the development of broad-spectrum antivirals.

The recognition of PPxY viral Late domains by the third WW domain of the human HECT-E3 ubiquitin ligase NEDD4 (NEDD4-WW3) is essential for the budding of many viruses. Blocking these interactions is a promising strategy to develop broad-spectrum antivirals. As all WW domains, NEDD4-WW3 is a challenging therapeutic target due to the low binding affinity of its natural interactions, its high conformational plasticity, and its complex thermodynamic behavior. In this work, we set out to investigate whether high affinity can be achieved for monovalent ligands binding to the isolated NEDD4-WW3 domain. We show that a competitive phage-display set-up allows for the identification of high-affinity peptides showing inhibitory activity of viral budding. A detailed biophysical study combining calorimetry, nuclear magnetic resonance, and molecular dynamic simulations reveals that the improvement in binding affinity does not arise from the establishment of new interactions with the domain, but is associated to conformational restrictions imposed by a novel C-terminal -LFP motif in the ligand, unprecedented in the PPxY interactome. These results, which highlight the complexity of WW domain interactions, provide valuable insight into the key elements for high binding affinity, of interest to guide virtual screening campaigns for the identification of novel therapeutics targeting NEDD4-WW3 interactions.

A Thermodynamic Analysis of the Binding Specificity between Four Human PDZ Domains and Eight Host, Viral and Designed Ligands.

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

The Conformational Plasticity Vista of PDZ Domains.

The PDZ domain (PSD95-Discs large-ZO1) is a widespread modular domain present in the living organisms. A prevalent function in the PDZ family is to serve as scaffolding and adaptor proteins connecting multiple partners in signaling pathways. An explanation of the flexible functionality in this domain family, based just on a static perspective of the structure-activity relationship, might fall short. More dynamic and conformational aspects in the protein fold can be the reasons for such functionality. Folding studies indeed showed an ample and malleable folding landscape for PDZ domains where multiple intermediate states were experimentally detected. Allosteric phenomena that resemble energetic coupling between residues have also been found in PDZ domains. Additionally, several PDZ domains are modulated by post-translational modifications, which introduce conformational switches that affect binding. Altogether, the ability to connect diverse partners might arise from the intrinsic plasticity of the PDZ fold.

PDZ/PDZ interaction between PSD-95 and nNOS neuronal proteins: A thermodynamic analysis of the PSD95-PDZ2/nNOS-PDZ interaction.

N-Methyl-D-aspartate (NMDA) receptors are key components in synaptic communication and are highly relevant in central nervous disorders, where they trigger excessive calcium entry into the neuronal cells causing harmful overproduction of nitric oxide by the neuronal nitric oxide synthase (nNOS) protein. Remarkably, NMDA receptor activation is aided by a second protein, postsynaptic density of 95 kDa (PSD95), forming the ternary protein complex NMDA/PSD95/nNOS. To minimize the potential side effects derived from blocking this ternary complex or either of its protein components, a promising approach points to the disruption of the PSD-95/nNOS interaction which is mediated by a PDZ/PDZ domain complex. Since the rational development of molecules targeting such protein-protein interaction relies on energetic and structural information herein, we include a thermodynamic and structural analysis of the PSD95-PDZ2/nNOS-PDZ. Two energetically relevant events are structurally linked to a "two-faced" or two areas of recognition between both domains. First, the assembly of a four-stranded antiparallel ß-sheet between the ß hairpins of nNOS and of PSD95-PDZ2, mainly enthalpic in nature, contributes 80% to the affinity. Second, binding is entropically reinforced by the hydrophobic interaction between side chains of the same nNOS ß-hairpin with the side chains of α2-helix at the binding site of PSD95-PDZ2, contributing the remaining 20% of the total affinity. These results suggest strategies for the future rational design of molecules able to disrupt this complex and constitute the first exhaustive thermodynamic analysis of a PDZ/PDZ interaction.

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1.

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.

Conformational changes in the third PDZ domain of the neuronal postsynaptic density protein 95.

PDZ domains are protein-protein recognition modules that interact with other proteins through short sequences at the carboxyl terminus. These domains are structurally characterized by a conserved fold composed of six ß-strands and two α-helices. The third PDZ domain of the neuronal postsynaptic density protein 95 has an additional α-helix (α3), the role of which is not well known. In previous structures, a succinimide was identified in the ß2-ß3 loop instead of Asp332. The presence of this modified residue results in conformational changes in α3. In this work, crystallographic structures of the following have been solved: a truncated form of the third PDZ domain of the neuronal postsynaptic density protein 95 from which α3 has been removed, D332P and D332G variants of the protein, and a new crystal form of this domain showing the binding of Asp332 to the carboxylate-binding site of a symmetry-related molecule. Crystals of the wild type and variants were obtained in different space groups, which reflects the conformational plasticity of the domain. Indeed, the overall analysis of these structures suggests that the conformation of the ß2-ß3 loop is correlated with the fold acquired by α3. The alternate conformation of the ß2-ß3 loop affects the electrostatics of the carboxylate-binding site and might modulate the binding of different PDZ-binding motifs.

The p12 subunit of human polymerase δ uses an atypical PIP box for molecular recognition of proliferating cell nuclear antigen (PCNA).

Human DNA polymerase δ is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). In addition to its catalytic subunit (p125), pol δ comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR-, isothermal calorimetry-, and X-ray crystallography-based analyses of the p12-PCNA interaction, which takes part in the modulation of the rate and fidelity of DNA synthesis by pol δ. We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP box located at the p12 N terminus. Unlike the canonical PIP box of p68, the PIP box of p12 lacks the conserved glutamine; binds through a 2-fork plug made of an isoleucine and a tyrosine residue at +3 and +8 positions, respectively; and is stabilized by an aspartate at +6 position, which creates a network of intramolecular hydrogen bonds. These findings add to growing evidence that PCNA can bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested.

Towards the improvement in stability of an anti-Aß single-chain variable fragment, scFv-h3D6, as a way to enhance its therapeutic potential.

ScFv-h3D6 is a single-chain variable fragment derived from the monoclonal antibody bapineuzumab that prevents Aß-induced cytotoxicity by capturing Aß oligomers. The benefits of scFv-h3D6 treatment in Alzheimer's disease are known at the behavioural, cellular and molecular levels in the 3xTg-AD mouse model. Antibody-based therapeutics are only stable in a limited temperature range, so their benefit in vivo depends on their capability for maintaining the proper fold. Here, we have stabilized the scFv-h3D6 folding by introducing the mutation VH-K64R and combining it with the previously described elongation of the VL domain (C3). The stabilities of the different scFv-h3D6 constructs were calculated from urea and thermal denaturation followed by Trp-fluorescence, CD and DSC and resulted in the order C3 > K64R/C3 > VH-K64R ≥ scFv-h3D6; showing that the combination of both mutations was not additive, instead they partially cancelled each other. The three mutants assayed showed a decreased aggregation tendency but maintained their capability to aggregate in the form of worm-like fibrils, basis of the protective effect of scFv-h3D6. Cytotoxicity assays showed that all the mutants recovered cell viability of Aß-treated neuroblastoma cell cultures in a dose-dependent manner and with efficiencies that correlated with stability, therefore improving the therapeutic ability of this antibody.

Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues.

Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogues directly from serine (Ser) and the corresponding indole analogue. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogues. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which aids in unlocking the synthetic potential of TrpB.

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity.

Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of l-tryptophan from l-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with l-threonine (Thr), leading to (2S,3S)-ß-methyltryptophan. Surprisingly, ß-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite a >82000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site, and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.

A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation.

Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase ß-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations.

Common features in the unfolding and misfolding of PDZ domains and beyond: the modulatory effect of domain swapping and extra-elements.

PDZ domains are protein-protein interaction modules sharing the same structural arrangement. To discern whether they display common features in their unfolding/misfolding behaviour we have analyzed in this work the unfolding thermodynamics, together with the misfolding kinetics, of the PDZ fold using three archetypical examples: the second and third PDZ domains of the PSD95 protein and the Erbin PDZ domain. Results showed that all domains passed through a common intermediate, which populated upon unfolding, and that this in turn drove the misfolding towards worm-like fibrillar structures. Thus, the unfolding/misfolding behaviour appears to be shared within these domains. We have also analyzed how this landscape can be modified upon the inclusion of extra-elements, as it is in the nNOS PDZ domain, or the organization of swapped species, as happens in the second PDZ domain of the ZO2 protein. Although the intermediates still formed upon thermal unfolding, the misfolding was prevented to varying degrees.

Directed evolution of the tryptophan synthase ß-subunit for stand-alone function recapitulates allosteric activation.

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the ß-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αßßα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.

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.

The impact of extra-domain structures and post-translational modifications in the folding/misfolding behaviour of the third PDZ domain of MAGUK neuronal protein PSD-95.

The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the ß2-ß3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two ß-sheets present in the PDZ3 fold. However, those mutations affecting the ß2-ß3 loop, included into the prone-to-aggregation region composed by a single ß-sheet comprising ß2 to ß4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.

Post-translational modifications modulate ligand recognition by the third PDZ domain of the MAGUK protein PSD-95.

The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the ß2-ß3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

A thermodynamic study of the third PDZ domain of MAGUK neuronal protein PSD-95 reveals a complex three-state folding behavior.

The relevance of the C-terminal α helix of the PDZ3 domain of PSD95 in its unfolding process has been explored by achieving the thermodynamic characterization of a construct where the sequence of the nine residues corresponding to such motif has been deleted. Calorimetric traces at neutral pH require the application of a three-state model displaying three different equilibrium processes in which the intermediate state self-associates upon heating, being stable and populated in a wide temperature range. Temperature scans followed by circular dichroism, Fourier transform infrared spectroscopy and dynamic light scattering support the presence of such oligomeric-partially folded species. This study reveals that the deletion of the α3-helix sequence results in a more complex description of the domain unfolding.