Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

A biophysical and structural analysis of the interaction of BLNK with 14-3-3 proteins.

B-cell linker protein (BLNK) is an adaptor protein that orchestrates signalling downstream of B-cell receptors. It has been reported to undergo proteasomal degradation upon binding to 14-3-3 proteins. Here, we report the first biophysical and structural study of this protein-protein interaction (PPI). Specifically, we investigated the binding of mono- and di- phosphorylated BLNK peptides to 14-3-3 using fluorescent polarization (FP) and isothermal titration calorimetry assays (ITC). Our results suggest that BLNK interacts with 14-3-3 according to the gatekeeper model, where HPK1 mediated phosphorylation of Thr152 (pT152) allows BLNK anchoring to 14-3-3, and an additional phosphorylation of Ser285 (pS285) by AKT, then further improves the affinity. Finally, we have also solved a crystal structure of the BLNKpT152 peptide bound to 14-3-3σ. These findings could serve as important tool for compound discovery programs aiming to modulate this interaction with 14-3-3.

Site-Directed Fragment-Based Screening for the Discovery of Protein-Protein Interaction Stabilizers.

Modulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.

Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.

Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD.

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.

Identification of Two Secondary Ligand Binding Sites in 14-3-3 Proteins Using Fragment Screening.

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein-protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation.

Designed Spiroketal Protein Modulation.

Spiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure-based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co-activator recruitment. We solved the crystal structure of the spiroketal-hRXRα-TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co-crystal structure, the first of a designed spiroketal-protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes.

The structure of the proteinaceous inhibitor PliI from Aeromonas hydrophila in complex with its target lysozyme.

Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Šresolution crystal structure of the periplasmic i-type lysozyme inhibitor from Aeromonas hydrophila (PliI-Ah) in complex with the i-type lysozyme from Meretrix lusoria is reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key-lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme-inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key-lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs.

Accounting for observed small angle X-ray scattering profile in the protein-protein docking server ClusPro.

The protein-protein docking server ClusPro is used by thousands of laboratories, and models built by the server have been reported in over 300 publications. Although the structures generated by the docking include near-native ones for many proteins, selecting the best model is difficult due to the uncertainty in scoring. Small angle X-ray scattering (SAXS) is an experimental technique for obtaining low resolution structural information in solution. While not sufficient on its own to uniquely predict complex structures, accounting for SAXS data improves the ranking of models and facilitates the identification of the most accurate structure. Although SAXS profiles are currently available only for a small number of complexes, due to its simplicity the method is becoming increasingly popular. Since combining docking with SAXS experiments will provide a viable strategy for fairly high-throughput determination of protein complex structures, the option of using SAXS restraints is added to the ClusPro server. © 2015 Wiley Periodicals, Inc.

Stabilizer-Guided Inhibition of Protein-Protein Interactions.

The discovery of novel protein-protein interaction (PPI) modulators represents one of the great molecular challenges of the modern era. PPIs can be modulated by either inhibitor or stabilizer compounds, which target different though proximal regions of the protein interface. In principle, protein-stabilizer complexes can guide the design of PPI inhibitors (and vice versa). In the present work, we combine X-ray crystallographic data from both stabilizer and inhibitor co-crystal complexes of the adapter protein 14-3-3 to characterize, down to the atomic scale, inhibitors of the 14-3-3/Tau PPI, a potential drug target to treat Alzheimer's disease. The most potent compound notably inhibited the binding of phosphorylated full-length Tau to 14-3-3 according to NMR spectroscopy studies. Our work sets a precedent for the rational design of PPI inhibitors guided by PPI stabilizer-protein complexes while potentially enabling access to new synthetically tractable stabilizers of 14-3-3 and other PPIs.

A natural-product switch for a dynamic protein interface.

Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand-binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and X-ray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-of-kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.

Chimeric antigens displaying GPR65 extracellular loops on a soluble scaffold enabled the discovery of antibodies, which recognized native receptor.

GPR65 is a proton-sensing G-protein coupled receptor associated with multiple immune-mediated inflammatory diseases, whose function is relatively poorly understood. With few reagents commercially available to probe the biology of receptor, generation of an anti-GPR65 monoclonal antibody was desired. Using soluble chimeric scaffolds, such as ApoE3, displaying the extracellular loops of GPR65, together with established phage display technology, native GPR65 loop-specific antibodies were identified. Phage-derived loop-binding antibodies recognized the wild-type native receptor to which they had not previously been exposed, generating confidence in the use of chimeric soluble proteins to act as efficient surrogates for membrane protein extracellular loop antigens. This technique provides promise for the rational design of chimeric antigens in facilitating the discovery of specific antibodies to GPCRs.

This technique offers a viable approach for antibody discovery to difficult GPCRs.Structurally relevant, soluble chimeric scaffold proteins of GPR65 were generated.Chimeric antigens were used to identify GPR65-specific antibodies by phage display.

Structural characterization of the PliG lysozyme inhibitor family.

Several Gram-negative bacteria protect themselves against the lytic action of host lysozymes by producing specific proteinaceous inhibitors. So far, four different families of lysozyme inhibitors have been identified including Ivy (Inhibitor of vertebrate lysozyme), MliC/PliC (Membrane associated/periplasmic inhibitor of C-type lysozyme), PliI and PliG (periplasmic inhibitors of I- and G-type lysozymes, respectively). Here we provide the first crystallographic description of the PliG family. Crystal structures were obtained for the PliG homologues from Escherichia coli, Salmonella enterica serotype Typhimurium and Aeromonas hydrophila. These structures show that the fold of the PliG family is very distinct from that of all other families of lysozyme inhibitors. Small-angle X-ray scattering studies reveal that PliG is monomeric in solution as opposed to the dimeric PliC and PliI. The PliG family shares a highly conserved SG(x)xY sequence motif with the MliC/PliC and PliI families where it was shown to reside on a loop that blocks the active site of lysozyme leading to inhibition. Surprisingly, we found that in PliG this motif is not well exposed and not involved in the inhibitory action. Instead, we could identify a distinct cluster of surface residues that are conserved across the PliG family and are essential for efficient G-type lysozyme inhibition, as evidenced by mutagenesis studies.

Molecular glues to stabilise protein-protein interactions.

Targeting protein-protein interactions (PPIs) has become a common approach to tackle various diseases whose pathobiology is driven by their mis-regulation in important signalling pathways. Modulating PPIs has tremendous untapped therapeutic potential and different approaches can be used to modulate PPIs. Initially, therapeutic effects were mostly sought by inhibiting PPIs. However, by gaining insight in the mode of action of certain therapeutic compounds, it became clear that stabilising (i.e. enhancing) PPIs can also be useful. The latter strategy is recently gaining a lot of attention, as stabilising physiologic, or even inducing novel interactions of a target protein with E3 ubiquitin ligases forms the basis of the targeted protein degradation (TPD) approach. An emerging additional example for drug discovery based on PPI stabilisation are the 14-3-3 proteins, a family of regulatory proteins, which engages in many protein-protein interactions, some of which might become therapeutical targets.

The identification and structural analysis of potential 14-3-3 interaction sites on the bone regulator protein Schnurri-3.

14-3-3 proteins regulate many intracellular processes and their ability to bind in subtly different fashions to their numerous partner proteins provides attractive drug-targeting points for a range of diseases. Schnurri-3 is a suppressor of mouse bone formation and a candidate target for novel osteoporosis therapeutics, and thus it is of interest to determine whether it interacts with 14-3-3. In this work, potential 14-3-3 interaction sites on mammalian Schnurri-3 were identified by an in silico analysis of its protein sequence. Using fluorescence polarization, isothermal titration calorimetry and X-ray crystallography, it is shown that synthetic peptides containing either phosphorylated Thr869 or Ser542 can indeed interact with 14-3-3, with the latter capable of forming an interprotein disulfide bond with 14-3-3σ: a hitherto unreported phenomenon.

The 14-3-3/SLP76 protein-protein interaction in T-cell receptor signalling: a structural and biophysical characterization.

The SH2 domain-containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T-cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14-3-3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14-3-3/SLP76 protein-protein interaction (PPI), we have determined a high-resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14-3-3σ. We then characterized its binding to 14-3-3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14-3-3. Our work lays the foundation for drug design efforts aimed at targeting the 14-3-3/SLP76 interaction and, thereby, TCR signalling.

Identification of molecular glues of the SLP76/14-3-3 protein-protein interaction.

The stabilisation of protein-protein interactions (PPIs) through molecular glues is a novel and promising approach in drug discovery. In stark contrast to research in protein-protein inhibition the field of stabilisation remains underdeveloped with comparatively few examples of small-molecule stabilisers of PPIs reported to date. At the same time identifying molecular glues has received recent sustained interest, especially in the fields of targeted protein degradation and 14-3-3 PPIs. The hub-protein 14-3-3 has a broad interactome with more than 500 known protein partners which presents a great opportunity for therapeutic intervention. In this study we have developed an HTRF assay suitable for HTS of the 14-3-3/SLP76 PPI and have completed a proof of concept screen against a chemically diverse library of 20 K molecules. The adaptor protein SLP76 has been reported to interact with 14-3-3 proteins downstream of the TCR playing an important role in mediating its own proteasomal degradation. We believe that stabilisation of this PPI could be exploited to potentiate degradation of SLP76 and therefore inhibit TCR signalling. This would represent an interesting alternative to other approaches in the field of targeted protein degradation. Here we disclose 16 novel stabilisers of the 14-3-3/SLP76 PPI across multiple different chemotypes. Based on the early results presented here we would recommend this approach to find molecular glues with broad applicability in the field of 14-3-3 PPIs.

A Structural Study of the Cytoplasmic Chaperone Effect of 14-3-3 Proteins on Ataxin-1.

Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or "chaperone" effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology.

Ligand-Based Design of Allosteric Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.

Fragment-based Differential Targeting of PPI Stabilizer Interfaces.

Stabilization of protein-protein interactions (PPIs) holds great potential for therapeutic agents, as illustrated by the successful drugs rapamycin and lenalidomide. However, how such interface-binding molecules can be created in a rational, bottom-up manner is a largely unanswered question. We report here how a fragment-based approach can be used to identify chemical starting points for the development of small-molecule stabilizers that differentiate between two different PPI interfaces of the adapter protein 14-3-3. The fragments discriminately bind to the interface of 14-3-3 with the recognition motif of either the tumor suppressor protein p53 or the oncogenic transcription factor TAZ. This X-ray crystallography driven study shows that the rim of the interface of individual 14-3-3 complexes can be targeted in a differential manner with fragments that represent promising starting points for the development of specific 14-3-3 PPI stabilizers.