TGIF1 homeodomain interacts with Smad MH1 domain and represses TGF-ß signaling.

TGIF1 is a multifunctional protein that represses TGF-ß-activated transcription by interacting with Smad2-Smad4 complexes. We found that the complex structure of TGIF1-HD bound to the TGACA motif revealed a combined binding mode that involves the HD core and the major groove, on the one hand, and the amino-terminal (N-term) arm and the minor groove of the DNA, on the other. We also show that TGIF1-HD interacts with the MH1 domain of Smad proteins, thereby indicating that TGIF1-HD is also a protein-binding domain. Moreover, the formation of the HD-MH1 complex partially hinders the DNA-binding site of the complex, preventing the efficient interaction of TGIF1-HD with DNA. We propose that the binding of the TGIF1 C-term to the Smad2-MH2 domain brings both the HD and MH1 domain into close proximity. This local proximity facilitates the interaction of these DNA-binding domains, thus strengthening the formation of the protein complex versus DNA binding. Once the protein complex has been formed, the TGIF1-Smad system would be released from promoters/enhancers, thereby illustrating one of the mechanisms used by TGIF1 to exert its function as an active repressor of Smad-induced TGF-ß signaling.

Structural determinants of Smad function in TGF-ß signaling.

Smad transcription factors are central to the signal transduction pathway that mediates the numerous effects of the transforming growth factor ß (TGF-ß) superfamily of cytokines in metazoan embryo development as well as in adult tissue regeneration and homeostasis. Although Smad proteins are conserved, recent genome-sequencing projects have revealed their sequence variation in metazoan evolution, human polymorphisms, and cancer. Structural studies of Smads bound to partner proteins and target DNA provide a framework for understanding the significance of these evolutionary and pathologic sequence variations. We synthesize the extant mutational and structural data to suggest how genetic variation in Smads may affect the structure, regulation, and function of these proteins. We also present a web application that compares Smad sequences and displays Smad protein structures and their disease-associated variants.

Preventing fibril formation of a protein by selective mutation.

The origins of formation of an intermediate state involved in amyloid formation and ways to prevent it are illustrated with the example of the Formin binding protein 28 (FBP28) WW domain, which folds with biphasic kinetics. Molecular dynamics of protein folding trajectories are used to examine local and global motions and the time dependence of formation of contacts between C(α)s and C(ß)s of selected pairs of residues. Focus is placed on the WT FBP28 WW domain and its six mutants (L26D, L26E, L26W, E27Y, T29D, and T29Y), which have structures that are determined by high-resolution NMR spectroscopy. The origins of formation of an intermediate state are elucidated, viz. as formation of hairpin 1 by a hydrophobic collapse mechanism causing significant delay of formation of both hairpins, especially hairpin 2, which facilitates the emergence of an intermediate state. It seems that three-state folding is a major folding scenario for all six mutants and WT. Additionally, two-state and downhill folding scenarios were identified in ∼ 15% of the folding trajectories for L26D and L26W, in which both hairpins are formed by the Matheson-Scheraga mechanism much faster than in three-state folding. These results indicate that formation of hairpins connecting two antiparallel ß-strands determines overall folding. The correlations between the local and global motions identified for all folding trajectories lead to the identification of the residues making the main contributions in the formation of the intermediate state. The presented findings may provide an understanding of protein folding intermediates in general and lead to a procedure for their prevention.

A tetradecapeptide somatostatin dicarba-analog: Synthesis, structural impact and biological activity.

We described here the first tetradecapeptide somatostatin-analogue where the disulfide bridge has been replaced by a carbon-carbon double bond. This analogue was prepared using microwave assisted ring closing metathesis (RCM) using the 2nd generation Grubbs as catalyst. Under our optimized conditions the cyclization between allylGly 3 and 14 proceeded in moderate yield, excellent cyclic/linear ratio and very high Z-double bond selectivity. NMR studies also demonstrated that the conformational flexibility of this peptide is increased in comparison to that of the natural hormone. Remarkably, this alkene-bridged somatostatin analog is highly selective against somatostatin receptors 1 and 5, suggesting that conformational rigidity is not required for the efficient interaction of somatostatin analogues with these two receptors.

First Generic Teriparatide: Structural and Biological Sameness to Its Reference Medicinal Product.

Teriparatide is an anabolic peptide drug indicated for the treatment of osteoporosis. Recombinant teriparatide was first approved in 2002 and has since been followed by patent-free alternatives under biosimilar or hybrid regulatory application. The aim of this study is to demonstrate the essential similarity between synthetic teriparatide BGW and the reference medicinal product (RMP), and thus to ensure the development of the first generic teriparatide drug. Hence, an extensive side-by-side comparative exercise, focusing on structural and biological activity, was performed using a wide range of state-of-the-art orthogonal methods. Nuclear magnetic resonance (NMR), ion mobility-mass spectrometry (IM-MS), UV, circular dichroism (CD) and Fourier transform infrared (FTIR) demonstrated the structural similarity between teriparatide BGW and the RMP. Comparative cell-based bioassays showed that the synthetic and recombinant peptides have identical behaviors. Teriparatide BGW, as a generic drug, provides an available treatment option for patients with osteoporosis and offers clinical benefits identical to those provided by the RMP.

Insights into structure-activity relationships of somatostatin analogs containing mesitylalanine.

The non-natural amino acid mesitylalanine (2,4,6-trimethyl-L-phenylalanine; Msa) has an electron-richer and a more conformationally restricted side-chain than that of its natural phenylalanine counterpart. Taking these properties into account, we have synthesized ten somatostatin analogs containing Msa residues in different key positions to modify the intrinsic conformational flexibility of the natural hormone. We have measured the binding affinity of these analogs and correlated it with the main conformations they populate in solution. NMR and computational analysis revealed that analogs containing one Msa residue were conformationally more restricted than somatostatin under similar experimental conditions. Furthermore, we were able to characterize the presence of a hairpin at the pharmacophore region and a non-covalent interaction between aromatic residues 6 and 11. In all cases, the inclusion of a D-Trp in the eighth position further stabilized the main conformation. Some of these peptides bound selectively to one or two somatostatin receptors with similar or even higher affinity than the natural hormone. However, we also found that multiple incorporations of Msa residues increased the life span of the peptides in serum but with a loss of conformational rigidity and binding affinity.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α.

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Characterization of p38α autophosphorylation inhibitors that target the non-canonical activation pathway.

p38α is a versatile protein kinase that can control numerous processes and plays important roles in the cellular responses to stress. Dysregulation of p38α signaling has been linked to several diseases including inflammation, immune disorders and cancer, suggesting that targeting p38α could be therapeutically beneficial. Over the last two decades, numerous p38α inhibitors have been developed, which showed promising effects in pre-clinical studies but results from clinical trials have been disappointing, fueling the interest in the generation of alternative mechanisms of p38α modulation. Here, we report the in silico identification of compounds that we refer to as non-canonical p38α inhibitors (NC-p38i). By combining biochemical and structural analyses, we show that NC-p38i efficiently inhibit p38α autophosphorylation but weakly affect the activity of the canonical pathway. Our results demonstrate how the structural plasticity of p38α can be leveraged to develop therapeutic opportunities targeting a subset of the functions regulated by this pathway.

HTSDSF Explorer, A Novel Tool to Analyze High-throughput DSF Screenings.

The identification of new drugs for novel therapeutic targets requires the screening of libraries containing tens of thousands of compounds. While experimental screenings are assisted by high-throughput technologies, in target-based biophysical assays, such as differential scanning fluorimetry (DSF), the analysis steps must be calculated manually, often combining several software packages. To simplify the determination of the melting temperature (Tm) of the target and the change induced by ligand binding (ΔTm), we developed the HTSDSF explorer, a versatile, all-in-one, user-friendly application suite. Implemented as a server-client application, in the primary screenings, HTSDSF explorer pre-analyzes and displays the Tm and ΔTm results interactively, thereby allowing the user to study hundreds of conditions and select the primary hits in minutes. This application also allows the determination of preliminary binding constants (KD) through a series of subsequent dose-response assays on the primary hits, thereby facilitating the ranking of validated hits and the advance of drug discovery efforts.

Conformational ensemble of the TNF-derived peptide solnatide in solution.

Tumor necrosis factor (TNF) is a homotrimer that has two spatially distinct binding regions, three lectin-like domains (LLD) at the TIP of the protein and three basolaterally located receptor-binding sites, the latter of which are responsible for the inflammatory and cell death-inducing properties of the cytokine. Solnatide (a.k.a. TIP peptide, AP301) is a 17-mer cyclic peptide that mimics the LLD of human TNF which activates the amiloride-sensitive epithelial sodium channel (ENaC) and, as such, recapitulates the capacity of TNF to enhance alveolar fluid clearance, as demonstrated in numerous preclinical studies. TNF and solnatide interact with glycoproteins and these interactions are necessary for their trypanolytic and ENaC-activating activities. In view of the crucial role of ENaC in lung liquid clearance, solnatide is currently being evaluated as a novel therapeutic agent to treat pulmonary edema in patients with moderate-to-severe acute respiratory distress syndrome (ARDS), as well as severe COVID-19 patients with ARDS. To facilitate the description of the functional properties of solnatide in detail, as well as to further target-docking studies, we have analyzed its folding properties by NMR. In solution, solnatide populates a set of conformations characterized by a small hydrophobic core and two electrostatically charged poles. Using the structural information determined here and also that available for the ENaC protein, we propose a model to describe solnatide interaction with the C-terminal domain of the ENaCα subunit. This model may serve to guide future experiments to validate specific interactions with ENaCα and the design of new solnatide analogs with unexplored functionalities.

Molecular basis for DNA recognition by the maternal pioneer transcription factor FoxH1.

Forkhead box H1 (FoxH1) is an essential maternal pioneer factor during embryonic development that binds to specific GG/GT-containing DNA target sequences. Here we have determined high-resolution structures of three FoxH1 proteins (from human, frog and fish species) and four DNAs to clarify the way in which FoxH1 binds to these sites. We found that the protein-DNA interactions extend to both the minor and major DNA grooves and are thus almost twice as extensive as those of other FOX family members. Moreover, we identified two specific amino acid changes in FoxH1 that allowed the recognition of GG/GT motifs. Consistent with the pioneer factor activity of FoxH1, we found that its affinity for nucleosomal DNA is even higher than for linear DNA fragments. The structures reported herein illustrate how FoxH1 binding to distinct DNA sites provides specificity and avoids cross-regulation by other FOX proteins that also operate during the maternal-zygotic transition and select canonical forkhead sites.

Controlling oncogenic KRAS signaling pathways with a Palladium-responsive peptide.

RAS oncoproteins are molecular switches associated with critical signaling pathways that regulate cell proliferation and differentiation. Mutations in the RAS family, mainly in the KRAS isoform, are responsible for some of the deadliest cancers, which has made this protein a major target in biomedical research. Here we demonstrate that a designed bis-histidine peptide derived from the αH helix of the cofactor SOS1 binds to KRAS with high affinity upon coordination to Pd(II). NMR spectroscopy and MD studies demonstrate that Pd(II) has a nucleating effect that facilitates the access to the bioactive α-helical conformation. The binding can be suppressed by an external metal chelator and recovered again by the addition of more Pd(II), making this system the first switchable KRAS binder, and demonstrates that folding-upon-binding mechanisms can operate in metal-nucleated peptides. In vitro experiments show that the metallopeptide can efficiently internalize into living cells and inhibit the MAPK kinase cascade.

SSTR1- and SSTR3-selective somatostatin analogues.

We prepared the two enantiomers of 3-(3'-quinolyl)-alanine (Qla, 1) in multigram scale by asymmetric hydrogenation. These amino acids, protected as Fmoc derivatives, were then used in the solid-phase synthesis of two new somatostatin 14 (SRIF-14) analogues 8 a and 8 b, tetradecapeptides in which the tryptophan residue (Trp8) is replaced by one of the two enantiomers of 3-(3'-quinolyl)-alanine (Qla8) and therefore lack the N--H bond in residue 8. The selectivity of these new analogues for the somatostatin receptors, SSTR1-5, was measured. Substitution with L-Qla8 yielded peptide 8 a, which was highly selective for SSTR1 and SSTR3, with an affinity similar to that of SRIF-14. Substitution by D-Qla gave the relatively selective analogue 8 b, which showed high affinity for SSTR3 and significant affinity for SSTR1, SSTR2 and SSTR5. The biological results demonstrate that bulky and electronically poor aromatic amino acids at position 8 are compatible with strong activity with SSTR1 and SSTR3. Remarkably, these high affinity levels were achieved with peptides in which the conformational mobility was increased with respect to that of SRIF-14. This observation suggests that conformational rigidity is not required, and might be detrimental to the interaction with receptors SSTR1 and SSTR3. The absence of an indole N proton in Qla8 might also contribute to the increased flexibility observed in these analogues.

Conformational landscape of multidomain SMAD proteins.

SMAD transcription factors, the main effectors of the TGFß (transforming growth factor ß) network, have a mixed architecture of globular domains and flexible linkers. Such a complicated architecture precluded the description of their full-length (FL) structure for many years. In this study, we unravel the structures of SMAD4 and SMAD2 proteins through an integrative approach combining Small-angle X-ray scattering, Nuclear Magnetic Resonance spectroscopy, X-ray, and computational modeling. We show that both proteins populate ensembles of conformations, with the globular domains tethered by disordered and flexible linkers, which defines a new dimension of regulation. The flexibility of the linkers facilitates DNA and protein binding and modulates the protein structure. Yet, SMAD4FL is monomeric, whereas SMAD2FL is in different monomer-dimer-trimer states, driven by interactions of the MH2 domains. Dimers are present regardless of the SMAD2FL activation state and concentration. Finally, we propose that SMAD2FL dimers are key building blocks for the quaternary structures of SMAD complexes.

Structures of the germline-specific Deadhead and thioredoxin T proteins from Drosophila melanogaster reveal unique features among thioredoxins.

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and thioredoxin T (TrxT), that belong to the lethal(3)malignant brain tumor signature genes and to the 'survival network' of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here, the first structures of Dhd and TrxT are presented, unveiling new features of these two thioredoxins. Dhd has positively charged patches on its surface, in contrast to the negatively charged surfaces commonly found in most Trxs. This distinctive charge distribution helps to define initial encounter complexes with DNA/RNA that will lead to final specific interactions with cofactors to promote chromatin remodeling. TrxT contains a C-terminal extension, which is mostly unstructured and highly flexible, that wraps the conserved core through a closed conformation. It is believed that these new structures can guide future work aimed at understanding embryo development and redox homeostasis in Drosophila. Moreover, due to their restricted presence in Schizophora (a section of the true flies), these structures can help in the design of small-molecular binders to modulate native redox homeostasis, thereby providing new applications for the control of plagues that cause human diseases and/or bring about economic losses by damaging crop production.

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes.

Smad transcription factors are the main downstream effectors of the Transforming growth factor ß superfamily (TGFß) signalling network. The DNA complexes determined here by X-ray crystallography for the Bone Morphogenetic Proteins (BMP) activated Smad5 and Smad8 proteins reveal that all MH1 domains bind [GGC(GC)|(CG)] motifs similarly, although TGFß-activated Smad2/3 and Smad4 MH1 domains bind as monomers whereas Smad1/5/8 form helix-swapped dimers. Dimers and monomers are also present in solution, as revealed by NMR. To decipher the characteristics that defined these dimers, we designed chimeric MH1 domains and characterized them using X-ray crystallography. We found that swapping the loop1 between TGFß- and BMP- activated MH1 domains switches the dimer/monomer propensities. When we scanned the distribution of Smad-bound motifs in ChIP-Seq peaks (Chromatin immunoprecipitation followed by high-throughput sequencing) in Smad-responsive genes, we observed specific site clustering and spacing depending on whether the peaks correspond to BMP- or TGFß-responsive genes. We also identified significant correlations between site distribution and monomer or dimer propensities. We propose that the MH1 monomer or dimer propensity of Smads contributes to the distinct motif selection genome-wide and together with the MH2 domain association, help define the composition of R-Smad/Smad4 trimeric complexes.

Structure-based design of a Cortistatin analogue with immunomodulatory activity in models of inflammatory bowel disease.

Ulcerative colitis and Crohn's disease are forms of inflammatory bowel disease whose incidence and prevalence are increasing worldwide. These diseases lead to chronic inflammation of the gastrointestinal tract as a result of an abnormal response of the immune system. Recent studies positioned Cortistatin, which shows low stability in plasma, as a candidate for IBD treatment. Here, using NMR structural information, we design five Cortistatin analogues adopting selected native Cortistatin conformations in solution. One of them, A5, preserves the anti-inflammatory and immunomodulatory activities of Cortistatin in vitro and in mouse models of the disease. Additionally, A5 displays an increased half-life in serum and a unique receptor binding profile, thereby overcoming the limitations of the native Cortistatin as a therapeutic agent. This study provides an efficient approach to the rational design of Cortistatin analogues and opens up new possibilities for the treatment of patients that fail to respond to other therapies.

Synthesis of Stable Cholesteryl-Polyethylene Glycol-Peptide Conjugates with Non-Disperse Polyethylene Glycol Lengths.

A method for conjugating cholesterol to peptide ligands through non-disperse polyethylene glycol (ND-PEG) through a non-hydrolysable linkage is described. The iterative addition of tetraethylene glycol macrocyclic sulfate to cholesterol (Chol) renders a family of highly pure well-defined Chol-PEG compounds with different PEG lengths from 4 up to 20 ethylene oxide units, stably linked through an ether bond. The conjugation of these Chol-PEG compounds to the cyclic (RGDfK) peptide though Lys5 side chains generates different lengths of Chol-PEG-RGD conjugates that retain the oligomer purity of the precursors, as analysis by HRMS and NMR has shown. Other derivatives were synthesized with similar results, such as Chol-PEG-OCH3 and Chol-PEG conjugated to glutathione and Tf1 peptides through maleimide-thiol chemoselective ligation. This method allows the systematic synthesis of highly pure uniform stable Chol-PEGs, circumventing the use of activation groups on each elongation step and thus reducing the number of synthesis steps.

NMR structural studies of the ItchWW3 domain reveal that phosphorylation at T30 inhibits the interaction with PPxY-containing ligands.

In this work, we study the role of phosphorylation as a regulatory mechanism for the interaction between the E3 ubiquitin ligase ItchWW3 domain and two PPxY motifs of one of its targets, the Epstein-Barr virus latent membrane protein 2A. Whereas ligand phosphorylation only diminishes binding, domain phosphorylation at residue T30 abrogates it. We show that two ItchWW domains can be phosphorylated at this position, using CK2 and PKA kinases and/or with stimulated T lymphocyte lysates. To better understand the regulation process, we determined the NMR structures of the ItchWW3-PPxY complex and of the phosphoT30-ItchWW3 variant. The peptide binds the domain using both XP and tyrosine grooves. A hydrogen bond from T30 to the ligand is also detected. This hydrogen-bond formation is precluded in the variant, explaining the inhibition upon phosphorylation. Our results suggest that phosphorylation at position 30 in ItchWW domains can be a mechanism to inhibit target recognition in vivo.

Binding site plasticity in viral PPxY Late domain recognition by the third WW domain of human NEDD4.

The recognition of PPxY viral Late domains by the third WW domain of the HECT-E3 ubiquitin ligase NEDD4 (hNEDD4-WW3) is essential for the completion of the budding process of numerous enveloped viruses, including Ebola, Marburg, HTLV1 or Rabies. hNEDD4-WW3 has been validated as a promising target for the development of novel host-oriented broad spectrum antivirals. 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. We present here a detailed structural and thermodynamic study of the interactions of hNEDD4-WW3 with viral Late domains combining isothermal titration calorimetry, NMR structural determination and molecular dynamics simulations. Structural and energetic differences in Late domain recognition reveal a highly plastic hNEDD4-WW3 binding site that can accommodate PPxY-containing ligands with varying orientations. These orientations are mostly determined by specific conformations adopted by residues I859 and T866. Our results suggest a conformational selection mechanism, extensive to other WW domains, and highlight the functional relevance of hNEDD4-WW3 domain conformational flexibility at the binding interface, which emerges as a key element to consider in the search for potent and selective inhibitors of therapeutic interest.