Eph receptors are involved in the regulation of cell adhesion and migration and are implicated in cancer progression, making them important drug targets. To date, the design of drugs targeting the ligand-binding domain of Eph includes the development of peptide mimetics of the ephrin ligands and the optimization of repurposed drugs. In this work, we report the results of a fragment-based screening (FBS) campaign against the ligand-binding domain of the EphA2 receptor. We introduce the workflow for the selection, filtering and follow-up studies of FBS hits, including an NMR/X-ray hybrid approach for the structure determination of protein/ligand complexes. The proposed workflow allowed us to identify several compounds with the receptor binding affinities of 50-100 µM and IC 50 of 1 µM , better than the activities of the known repurposed drugs. Due to the low molecular weight, the newly developed hits that exhibit an initial biological effect have a high potential for further optimization.

Objective: Reconstituting a membrane protein in a membrane-like environment is an essential factor, determining the relevance of structural data obtained for a membrane protein. Mixtures of lauryl maltose neopentyl glycol (LMNG) with cholesteryl hemisuccinate (CHS) are a recently introduced membrane-like medium with a proven stabilizing effect on membrane proteins of several classes. However, the structure of LMNG/CHS micelles remains underinvestigated. Methods: In the present work, we apply diffusion and nuclear Overhauser effect NMR spectroscopy and the transmembrane domain pf EphA2 as a sensor transmembrane protein to characterize the properties of LMNG/CHS micelles in comparison to other membrane mimetics, including phospholipid bicelles. Results and Discussion: According to the diffusion measurements, CHS addition to LMNG micelles reduces the temperature- and concentration-induced growth of the particles, preventing LMNG from forming non-regular rod-like structures. CHS is evenly distributed within the micelles, with no preference for the protein-adjacent regions. For a model protein, LMNG/CHS provides an environment similar to dodecyl maltoside but distinct from lipid bicelles in terms of lipid-protein contacts. Conclusions: LMNG/CHS is a membrane mimetic that stabilizes membrane proteins, has a defined micellar structure with even CHS distribution, suppresses undesirable rod-like LMNG aggregation, and offers a lipid-protein contact environment different from that of bicelles but similar to dodecyl maltoside.

Ephrin growth factors are attached to the membrane by either GPI-anchor or transmembrane domains and are known to signal bi-directionally: cells, expressing the ephrins experience the downstream signaling in response to ephrin recognition by their receptor, Eph. This bidirectionality makes ephrins an interesting drug target. In the present work, we investigate the role played by conformational heterogeneity and juxtamembrane domains of Ephrin A1 in its interactions with the ligand-binding domain of EphA2 receptor by NMR spectroscopy. We show that ligand recognition occurs via a conformational selection mechanism and that the juxtamembrane regions are flexible, unstructured and are not involved in the binding.

Acid-sensing ion channel 1a (ASIC1a) is involved in processes associated with fear, learning, and neurodegeneration within the central nervous system. However, ASIC1a is also abundant in the peripheral nervous system, where its role is still poorly understood, largely due to the lack of selective ligands. In this study, we present the discovery of the first selective positive allosteric modulator for ASIC1a, isolated from the sea anemone Metridium senile. The active compound, a peptide named Ms13-1, features a novel type of fold named 'Cys-ladder'. Ms13-1 exhibits high affinity and selectivity for ASIC1a, enhancing channel activation in response to a broad range of acidic stimuli (pH 6.9-5.5) without altering the proton affinity for the channel. Moreover, injection of Ms13-1 into the hind paw of mice provokes robust and long-lasting pain-related behavior, which is significantly attenuated by a selective ASIC1 antagonist. The discovery of this novel selective positive allosteric modulator opens up new perspectives to investigate the role of ASIC1a in various physiological processes.

Toll-like receptors (TLRs) are important players in the innate immune system. Binding of pathogen-related molecules to the extracellular domains of TLRs initiates signalosome assembly, a key event in signal transduction. Despite extensive research on individual receptor domains, the mechanism of signalosome assembly remains unclear. Recent evidence suggests that the intracellular TIR domain of TLR1 binds zinc ions, with cysteines playing a pivotal role in binding and receptor activation. This study explores the zinc-binding ability of the TLR2 TIR domain (TLR2TIR). We found that TLR2TIR binds zinc with nanomolar affinity through its cysteine residues. Two of them, C673 and C713, are essential for receptor activation. These results suggest that zinc may be involved in the initiation of signalosome assembly.

BACKGROUND: Diverse antidepressants were recently described to bind to TrkB (tyrosine kinase B) and drive a positive allosteric modulation of endogenous BDNF (brain-derived neurotrophic factor). Although neurotrophins such as BDNF can bind to p75NTR (p75 neurotrophin receptor), their precursors are the high-affinity p75NTR ligands. While part of an unrelated receptor family capable of inducing completely opposite physiological changes, TrkB and p75NTR feature a crosslike conformation dimer and carry a cholesterol-recognition amino acid consensus in the transmembrane domain. As such qualities were found to be crucial for antidepressants to bind to TrkB and drive behavioral and neuroplasticity effects, we hypothesized that their effects might also depend on p75NTR. METHODS: Enzyme-linked immunosorbent assay-based binding and nuclear magnetic resonance spectroscopy were performed to assess whether antidepressants would bind to p75NTR. HEK293T cells and a variety of in vitro assays were used to investigate whether fluoxetine (FLX) or ketamine (KET) would trigger any a- and g-secretase- dependent p75NTR proteolysis and lead to p75NTR nuclear localization. Ocular dominance shift was performed with male and female p75NTR knockout mice to study the effects of KET and FLX on brain plasticity, in addition to pharmacological interventions to verify how p75NTR signaling is important for the effects of KET and FLX in enhancing extinction memory in male wild-type mice and rats. RESULTS: Antidepressants were found to bind to p75NTR. FLX and KET triggered the p75NTR proteolytic pathway and induced p75NTR-dependent behavioral/neuroplasticity changes. CONCLUSIONS: We hypothesize that antidepressants co-opt both BDNF/TrkB and proBDNF/p75NTR systems to induce a more efficient activity-dependent synaptic competition, thereby boosting the brain's ability for remodeling.

SSR128129E (SSR) is a unique small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high-affinity allosteric binder that selectively blocks one of the two major FGFR-mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N-terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b-induced FGFR signaling compared to FGF2-induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N-terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water-soluble subdomains of the FGFR proteins points to the important role played by the cell membrane.

The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer–lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid–SMA-EA and lipid–SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy.

Objective: One of the major problems in the field of neurotrophin signaling is the role of Trk juxtamembrane regions. Here we present the production protocol of the d5 ligand-binding domain of TrkA with the full-length extracellular juxtamembrane region for structural studies. Methods: The protein was produced in E. coli cells. Protein purification included immobilized metal affinity and size-exclusion chromatography in the presence of urea. Refolding was performed using three approaches: dialysis, pulse and flash dilution. The quality of the final protein was assessed by gel filtration and NMR. Results and Discussion: We demonstrated that the obtained strain allows the production of milligram quantities of the target protein, including its isotope-labeled derivatives. A comparison of several refolding protocols revealed that dialysis and flash dilution are optimal, with the latter option being more economically feasible. Conclusions: Analysis of the final protein preparation showed that the proposed protein expression, purification, and refolding scheme allows the production of a highly purified protein suitable for structural and functional studies.

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling. A genetically encoded labeling system uses smallest-in-class fluorogen-activating protein tags for time-resolved fluorescence multiplexed cellular imaging, offering monoexponential decay and potential for sophisticated fluorescence lifetime analysis.

Puromycin (Puro) is a natural aminonucleoside antibiotic that inhibits protein synthesis by its incorporation into elongating peptide chains. The unique mechanism of Puro finds diverse applications in molecular biology, including the selection of genetically engineered cell lines, in situ protein synthesis monitoring, and studying ribosome functions. However, the key step of Puro biosynthesis remains enigmatic. In this work, pur6-guided genome mining is carried out to explore the natural diversity of Puro-like antibiotics. The diversity of biosynthetic gene cluster (BGC) architectures suggests the existence of distinct structural analogs of puromycin encoded by pur-like clusters. Moreover, the presence of tRNACys in some BGCs, i.e., cst-like clusters, leads us to the hypothesis that Pur6 utilizes aminoacylated tRNA as an activated peptidyl precursor, resulting in cysteine-based analogs. Detailed metabolomic analysis of Streptomyces sp. VKM Ac-502 containing cst-like BGC revealed the production of a cysteinyl-based analog of Puro—cystocin (Cst). Similar to puromycin, cystocin inhibits both prokaryotic and eukaryotic translation by the same mechanism. Aminonucleoside N-acetyltransferase CstC inactivated Cst, mediating antibiotic resistance in genetically modified bacteria and human cells. The substrate specificity of CstC originated from the steric hindrance of its active site. We believe that novel aminonucleosides and their inactivating enzymes can be developed through the directed evolution of the discovered biosynthetic machinery.

Transient receptor potential vanilloid subtype 3 (TRPV3) is an ion channel implicated in skin physiology and itch. TRPV3 inhibitors can present a novel strategy for combating debilitating itch conditions, and medicinal plants are a natural pool of such compounds. Here, we report the isolation of a TRPV3-inhibiting compound from Andrographis paniculata, a medicinal plant with anti-inflammatory properties whose bioactive components are poorly characterized in terms of molecular targets. Using 1H and 13C NMR and high-resolution mass spectrometry, the compound was identified as a labdane-type diterpenoid, 14-deoxy-11,12-didehydroandrographolide (ddA). The activity of the compound was evaluated by fluorescent calcium assay and manual whole-cell patch-clamp technique. ddA inhibited human TRPV3 in stably expressing CHO and HaCaT keratinocytes, acting selectively among other TRP channels implicated in itch and inflammation and not showing toxicity to HaCaT cells. Antipruritic effects of the compound were evaluated in scratching behavior models on ICR mice. ddA suppressed itch induced by the TRPV3 activator carvacrol. Additionally, ddA potently suppressed histamine-induced itch with efficacy comparable to loratadine, a clinically used antihistamine drug. These results suggest the potential of ddA as a possible safe and efficacious alternative for antipruritic therapy.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E. coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7TIR and MBP-tagged TLR3/5/7/8TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.

Neurotrophin receptors of the Trk family are involved in the regulation of brain development and neuroplasticity, and therefore can serve as targets for anti-cancer and stroke-recovery drugs, antidepressants, and many others. The structures of Trk protein domains in various states upon activation need to be elucidated to allow rational drug design. However, little is known about the conformations of the transmembrane and juxtamembrane domains of Trk receptors. In the present study, we employ NMR spectroscopy to solve the structure of the TrkB dimeric transmembrane domain in the lipid environment. We verify the structure using mutagenesis and confirm that the conformation corresponds to the active state of the receptor. Subsequent study of TrkB interaction with the antidepressant drug fluoxetine, and the antipsychotic drug chlorpromazine, provides a clear self-consistent model, describing the mechanism by which fluoxetine activates the receptor by binding to its transmembrane domain. Neurotrophin receptor TrkB regulates neuronal growth and neuroplasticity. Here, the authors present the NMR structure of the intramembrane region of TrkB activated by antidepressant drugs, yielding insights into receptor function.

Ephrin receptors regulate intercellular communication and are thus involved in tumor development. Ephrin receptor A2 (EphA2), in particular, is overexpressed in a variety of cancers and is a proven target for anti-cancer drugs. The N-terminal ligand-binding domain of ephrin receptors is responsible for the recognition of their ligands, ephrins, and is directly involved in receptor activation. Here, we report on the complete 1H, 15N and 13C NMR chemical shift assignment of EphA2 ligand binding domain that provides the basis for NMR-assisted drug design.

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation.

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol–water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol–water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains—transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.