Development of LM-41 and AF-2112, two flufenamic acid-derived TEAD inhibitors obtained through the replacement of the trifluoromethyl group by aryl rings.

The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.

Evidence of the Reduced Abundance of Proline cis Conformation in Protein Poly Proline Tracts.

Proline is found in a cis conformation in proteins more often than other proteinogenic amino acids, where it influences structure and modulates function, being the focus of several high-resolution structural studies. However, until now, technical and methodological limitations have hampered the site-specific investigation of the conformational preferences of prolines present in poly proline (poly-P) homorepeats in their protein context. Here, we apply site-specific isotopic labeling to obtain high-resolution NMR data on the cis/trans equilibrium of prolines within the poly-P repeats of huntingtin exon 1, the causative agent of Huntington's disease. Screening prolines in different positions in long (poly-P11) and short (poly-P3) poly-P tracts, we found that, while the first proline of poly-P tracts adopts similar levels of cis conformation as isolated prolines, a length-dependent reduced abundance of cis conformers is observed for terminal prolines. Interestingly, the cis isomer could not be detected in inner prolines, in line with percentages derived from a large database of proline-centered tripeptides extracted from crystallographic structures. These results suggest a strong cooperative effect within poly-Ps that enhances their stiffness by diminishing the stability of the cis conformation. This rigidity is key to rationalizing the protection toward aggregation that the poly-P tract confers to huntingtin. Furthermore, the study provides new avenues to probe the structural properties of poly-P tracts in protein design as scaffolds or nanoscale rulers.

Site-Specific Isotopic Labeling (SSIL): Access to High-Resolution Structural and Dynamic Information in Low-Complexity Proteins.

Remarkable technical progress in the area of structural biology has paved the way to study previously inaccessible targets. For example, large protein complexes can now be easily investigated by cryo-electron microscopy, and modern high-field NMR magnets have challenged the limits of high-resolution characterization of proteins in solution. However, the structural and dynamic characteristics of certain proteins with important functions still cannot be probed by conventional methods. These proteins in question contain low-complexity regions (LCRs), compositionally biased sequences where only a limited number of amino acids is repeated multiple times, which hamper their characterization. This Concept article describes a site-specific isotopic labeling (SSIL) strategy, which combines nonsense suppression and cell-free protein synthesis to overcome these limitations. An overview on how poly-glutamine tracts were made amenable to high-resolution structural studies is used to illustrate the usefulness of SSIL. Furthermore, we discuss the potential of this methodology to give further insights into the roles of LCRs in human pathologies and liquid-liquid phase separation, as well as the challenges that must be addressed in the future for the popularization of SSIL.

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats.

Homorepeat (HR) proteins are involved in key biological processes and multiple pathologies, however their high-resolution characterization has been impaired due to their homotypic nature. To overcome this problem, we have developed a strategy to isotopically label individual glutamines within HRs by combining nonsense suppression and cell-free expression. Our method has enabled the NMR investigation of huntingtin exon1 with a 16-residue polyglutamine (poly-Q) tract, and the results indicate the presence of an N-terminal α-helix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing a labeled glutamine into a pathological version of huntingtin with 46 glutamines. This methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly-Q-related diseases. Our approach can be extended to other amino acids to investigate biological processes involving proteins containing low-complexity regions (LCRs).

Epithelial Splicing Regulatory Protein 1 (ESRP1) is a new regulator of stomach smooth muscle development and plasticity.

In vertebrates, stomach smooth muscle development is a complex process that involves the tight transcriptional or post-transcriptional regulation of different signalling pathways. Here, we identified the RNA-binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) as an early marker of developing and undifferentiated stomach mesenchyme. Using a gain-of-function approach, we found that in chicken embryos, sustained expression of ESRP1 impairs stomach smooth muscle cell (SMC) differentiation and FGFR2 splicing profile. ESRP1 overexpression in primary differentiated stomach SMCs induced their dedifferentiation, promoted specific-FGFR2b splicing and decreased FGFR2c-dependent activity. Moreover, co-expression of ESRP1 and RBPMS2, another RNA-binding protein that regulates SMC plasticity and Bone Morphogenetic Protein (BMP) pathway inhibition, synergistically promoted SMC dedifferentiation. Finally, we also demonstrated that ESRP1 interacts with RBPMS2 and that RBPMS2-mediated SMC dedifferentiation requires ESRP1. Altogether, these results show that ESRP1 is expressed also in undifferentiated stomach mesenchyme and demonstrate its role in SMC development and plasticity.

Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome.

Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division.

Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39-51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

Two crystal structures of the FK506-binding domain of Plasmodium falciparum FKBP35 in complex with rapamycin at high resolution.

Antimalarial chemotherapy continues to be challenging in view of the emergence of drug resistance, especially artemisinin resistance in Southeast Asia. It is critical that novel antimalarial drugs are identified that inhibit new targets with unexplored mechanisms of action. It has been demonstrated that the immunosuppressive drug rapamycin, which is currently in clinical use to prevent organ-transplant rejection, has antimalarial effects. The Plasmodium falciparum target protein is PfFKBP35, a unique immunophilin FK506-binding protein (FKBP). This protein family binds rapamycin, FK506 and other immunosuppressive and non-immunosuppressive macrolactones. Here, two crystallographic structures of rapamycin in complex with the FK506-binding domain of PfFKBP35 at high resolution, in both its oxidized and reduced forms, are reported. In comparison with the human FKBP12-rapamycin complex reported previously, the structures reveal differences in the ß4-ß6 segment that lines the rapamycin binding site. Structural differences between the Plasmodium protein and human hFKBP12 include the replacement of Cys106 and Ser109 by His87 and Ile90, respectively. The proximity of Cys106 to the bound rapamycin molecule (4-5 Å) suggests possible routes for the rational design of analogues of rapamycin with specific antiparasitic activity. Comparison of the structures with the PfFKBD-FK506 complex shows that both drugs interact with the same binding-site residues. These two new structures highlight the structural differences and the specific interactions that must be kept in consideration for the rational design of rapamycin analogues with antimalarial activity that specifically bind to PfFKBP35 without immunosuppressive effects.

Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography.

X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries.

Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction.

Arrestin-dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR's C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three rhodopsin-like GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR), and the ß2-adernergic receptor (ß2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motifs (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transitions appear to favor arrestin-2 binding. Hence, our results suggest a model in which the phosphorylation-dependent structuration of the GPCR C-terminal regions would modulate arrestin binding and therefore signaling outcomes in arrestin-dependent pathways.

Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies.

Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems.

Multi-site-specific isotopic labeling accelerates high-resolution structural investigations of pathogenic huntingtin exon-1.

Huntington's disease neurodegeneration occurs when the number of consecutive glutamines in the huntingtin exon-1 (HTTExon1) exceeds a pathological threshold of 35. The sequence homogeneity of HTTExon1 reduces the signal dispersion in NMR spectra, hampering its structural characterization. By simultaneously introducing three isotopically labeled glutamines in a site-specific manner in multiple concatenated samples, 18 glutamines of a pathogenic HTTExon1 with 36 glutamines were unambiguously assigned. Chemical shift analyses indicate the α-helical persistence in the homorepeat and the absence of an emerging toxic conformation around the pathological threshold. Using the same type of samples, the recognition mechanism of Hsc70 molecular chaperone has been investigated, indicating that it binds to the N17 region of HTTExon1, inducing the partial unfolding of the poly-Q. The proposed strategy facilitates high-resolution structural and functional studies in low-complexity regions.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration.

The transcription factor YAP-TEAD is the downstream effector of the Hippo pathway which controls cell proliferation, apoptosis, tissue repair, and organ growth. Dysregulation of the Hippo pathway has been correlated with carcinogenic processes. A co-crystal structure of TEAD with its endogenous ligand palmitic acid (PA) as well as with flufenamic acid (FA) has been disclosed. Here we report the development of HC-258, which derives from FA and possesses an oxopentyl chain that mimics a molecule of PA as well as an acrylamide that reacts covalently with TEAD's cysteine. HC-258 reduces the CTGF, CYR61, AXL, and NF2 transcript levels and inhibits the migration of MDA-MB-231 breast cancer cells. Co-crystallization with hTEAD2 confirmed that HC-258 binds within TEAD's PA pocket, where it forms a covalent bond with its cysteine.

Toward the Design of Ligands Selective for the C-Terminal Domain of TEADs.

The Hippo signaling pathway plays a fundamental role in the control of organ growth, cell proliferation, and stem cell characters. TEADs are the main transcriptional output regulators of the Hippo signaling pathway and bind to YAP and TAZ co-activators. TEAD1-4 are expressed differently, depending on the tissue and developmental level, and can be overexpressed in certain pathologies. TEAD ligands mainly target the internal pocket of the C-terminal domain of TEAD, and the first ligands selective for TEAD1 and TEAD3 have been recently reported. In this paper, we focus on the topographic homology of the TEAD C-terminal domain both externally and in the internal pocket to highlight the possibility of rationally designing ligands selective for one of the TEAD family members. We identified a novel TEAD2-specific pocket and reported its first ligand. Finally, AlphaFold2 models of full-length TEADs suggest TEAD autoregulation and emphasize the importance of the interface 2.

Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction.

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the ß2-adernergic receptor (ß2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

A Flexible and Original Architecture of Two Unrelated Zinc Fingers Underlies the Role of the Multitask P1 in RYMV Spread.

Viruses of the sobemovirus genus are plant viruses, most of which generate very important agricultural and financial losses. Among them, the rice yellow mottle virus (RYMV) is one of the most damaging pathogens devastating rice fields in Africa. RYMV infectivity and propagation rely on its protein P1, identified as a key movement and potential long-distance RNA silencing suppressor. Here we describe P1's complete 3D structure and dynamics obtained by an integrative approach combining X-Ray crystallography and NMR spectroscopy. We show that P1 is organized in two semi-independent and topologically unrelated domains, each harboring an original zinc finger. The two domains exhibit different affinities for zinc and sensitivities to oxidoreduction conditions, making the C-terminal P1 region a potential labile sensor of the plant redox status. An additional level of regulation resides on the capacity of P1 to oligomerize through its N-terminal domain. Coupling P1 structure information with site-directed mutagenesis and plant functional assays, we identified key residues in each zinc domain essential for infectivity and spread in rice tissues. Altogether, our results provide the first complete structure of a sobemoviral P1 movement protein and highlight structural and dynamical properties that may serve RYMV functions to infect and invade its host plant.

SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo.

DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5'-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly.

An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test.

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR).

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.