The T Cell Antigen Receptor α Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations.

Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.

Computational inference of eIF4F complex function and structure in human cancers.

The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.

Malignant tumor cells engender second membrane-lined organelles for self-protection and tumor progression.

Cancer is a leading cause of mortality in humans, but the efficacy of current treatments for many cancers is limited, as they lack unique mechanistically defined targets. Here, we show that, upon malignant transformation, aggressive oncocells generate a second membrane exterior to their plasma membrane to form cytocapsulas (CCs) and cytocapsular tubes (CCTs), which all together constitute cytocapsular oncocells with pleotropic biological functions in cancer patient tissues in vivo. Proteomic and biochemical analyses revealed that the PMCA2 calcium pump is highly up-regulated in CCs and CCTs in malignant tumors but not in normal tissues, thus identifying a unique cancer biomarker and target for cancer therapy. Cytocapsular oncocells are universally present in solid cancers and appear in hematologic cancers in immune organs. Multi-cell malignant tumors are also enveloped by protective CC membranes. These cytocapsular tumors (CTs) generate numerous CCTs that form freeways for cancer cell metastasis to both neighboring and distant destinations. Entire cytocapsular tumor networks (CTNs) dominate physical cancer metastasis pathways in cancer patients in vivo. Later, CCTs invade micro blood vessels and release cytocapsular oncocells into the blood, providing a source of circulating tumor cells. CTNs interconnect cytocapsular tumors in primary and secondary cancer niches, creating larger cytocapsular tumor network systems (CTNSs). Primary and secondary CTNSs are in turn interconnected, forming dynamic and integrated CTNSs. Thus, interconnected cytocapsular oncocells, CTNs, and CTNSs coordinate cancer progression via the integrated cytocapsular membrane systems.

Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival.

Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the antiapoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We propose that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

An open-source drug discovery platform enables ultra-large virtual screens.

On average, an approved drug currently costs US$2-3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.

Challenges for chemistry in Ukraine after the war: Ukrainian science requires rebuilding and support.

The unprovoked Russian invasion has created considerable challenges for Ukrainian science. In this article, we discuss actions needed to support and rebuild Ukrainian science and educational systems. The proposed actions take into account past Ukrainian scientific achievements including developments in organic chemistry.

Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation.

The RNA helicase eIF4A plays a key role in unwinding of mRNA and scanning during translation initiation. Free eIF4A is a poor helicase and requires the accessory proteins eIF4G and eIF4H. However, the structure of the helicase complex and the mechanisms of stimulation of eIF4A activity have remained elusive. Here we report the topology of the eIF4A/4G/4H helicase complex, which is built from multiple experimentally observed domain-domain contacts. Remarkably, some of the interactions are continuously rearranged during the ATP binding/hydrolysis cycle of the helicase. We show that the accessory proteins modulate the affinity of eIF4A for ATP by interacting simultaneously with both helicase domains and promoting either the closed, ATP-bound conformation or the open, nucleotide-free conformation. The topology of the complex and the spatial arrangement of the RNA-binding surfaces offer insights into their roles in stimulation of helicase activity and the mechanisms of mRNA unwinding and scanning.

On the concept of systematization in the Kemeny-Oppenheim approach to intertheoretical reduction.

In 1956, John G. Kemeny and Paul Oppenheim proposed an approach to intertheoretical reduction as an alternative to that of Ernest Nagel. However, they neglected to provide a clear definition of its basic concept of systematization. After decades of languishing in the shadows, new interest in the KO approach is emerging. Nevertheless, there are still misunderstandings regarding this basic concept. The present paper elucidates this concept by returning to Oppenheim's hitherto little-noticed publications from the 1920s and 1930s, which Kemeny and Oppenheim obviously used as guidance in 1956. Reappraising Oppenheim's early writings delivers two significant payoffs: new clarity in understanding the concept of systematization as well as a more solid grasp of the structure of this approach as a distinctive combination of explanation and systematization.

NMR sample optimization and backbone assignment of a stabilized neurotensin receptor.

G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

Aromatic 19F-13C TROSY: a background-free approach to probe biomolecular structure, function, and dynamics.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.

A newly identified Leishmania IF4E-interacting protein, Leish4E-IP2, modulates the activity of cap-binding protein paralogs.

Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the preinitiation complex at the 5' end of mRNAs and regulates translation initiation. The requirement of Leishmania to survive in changing environments can explain why they encode multiple eIF4E (LeishIF4Es) and eIF4G (LeishIF4Gs) paralogs, as each could be assigned a discrete role during their life cycle. Here we show that the expression and activity of different LeishIF4Es change during the growth of cultured promastigotes, urging a search for regulatory proteins. We describe a novel LeishIF4E-interacting protein, Leish4E-IP2, which contains a conserved Y(X)4LΦ IF4E-binding-motif. Despite its capacity to bind several LeishIF4Es, Leish4E-IP2 was not detected in m7GTP-eluted cap-binding complexes, suggesting that it could inhibit the cap-binding activity of LeishIF4Es. Using a functional assay, we show that a recombinant form of Leish4E-IP2 inhibits the cap-binding activity of LeishIF4E-1 and LeishIF4E-3. Furthermore, we show that transgenic parasites expressing a tagged version of Leish4E-IP2 also display reduced cap-binding activities of tested LeishIF4Es, and decreased global translation. Given its ability to bind more than a single LeishIF4E, we suggest that Leish4E-IP2 could serve as a broad-range repressor of Leishmania protein synthesis.

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

Cytocapsular tubes conduct cell translocation.

Cell locomotion is essential for multicellular organism embryo development, organ homeostasis, tissue regeneration, immune responses, and tumor metastasis. Here we report that single mammalian cells can generate two extracellular membranous compartments: cytocapsulae and cytocapsular tubes. Cells migrate in cytocapsulae and engender cytocapsular tubes, which exhibit pleiotropic biological functions and provide tubular routes for directed cell transportation. Ultrastructural analysis by electron microscope revealed that nanoprotrusions surround and anchor cytocapsular tubes in place. Multiple cytocapsular tubes interconnect and form networks supporting directed cell transportation in diverse directions. Enhanced translation initiation factor eIF4E up-regulates translation of transcripts encoding proteins important for organelle development. Thus, this study proposes a mechanism of directed cell translocation in cytocapsular tubes, which may facilitate the management of diseases, including tumor metastasis.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins.

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

VirtualFlow Ants-Ultra-Large Virtual Screenings with Artificial Intelligence Driven Docking Algorithm Based on Ant Colony Optimization.

The docking program PLANTS, which is based on ant colony optimization (ACO) algorithm, has many advanced features for molecular docking. Among them are multiple scoring functions, the possibility to model explicit displaceable water molecules, and the inclusion of experimental constraints. Here, we add support of PLANTS to VirtualFlow (VirtualFlow Ants), which adds a valuable method for primary virtual screenings and rescoring procedures. Furthermore, we have added support of ligand libraries in the MOL2 format, as well as on the fly conversion of ligand libraries which are in the PDBQT format to the MOL2 format to endow VirtualFlow Ants with an increased flexibility regarding the ligand libraries. The on the fly conversion is carried out with Open Babel and the program SPORES. We applied VirtualFlow Ants to a test system involving KEAP1 on the Google Cloud up to 128,000 CPUs, and the observed scaling behavior is approximately linear. Furthermore, we have adjusted several central docking parameters of PLANTS (such as the speed parameter or the number of ants) and screened 10 million compounds for each of the 10 resulting docking scenarios. We analyzed their docking scores and average docking times, which are key factors in virtual screenings. The possibility of carrying out ultra-large virtual screening with PLANTS via VirtualFlow Ants opens new avenues in computational drug discovery.

The precious fluorine on the ring: fluorine NMR for biological systems.

The fluorine-19 nucleus was recognized early to harbor exceptional properties for NMR spectroscopy. With 100% natural abundance, a high gyromagnetic ratio (83% sensitivity compared to 1H), a chemical shift that is extremely sensitive to its surroundings and near total absence in biological systems, it was destined to become a favored NMR probe, decorating small and large molecules. However, after early excitement, where uptake of fluorinated aromatic amino acids was explored in a series of animal studies, 19F-NMR lost popularity, especially in large molecular weight systems, due to chemical shift anisotropy (CSA) induced line broadening at high magnetic fields. Recently, two orthogonal approaches, (i) CF3 labeling and (ii) aromatic 19F-13C labeling leveraging the TROSY (Transverse Relaxation Optimized Spectroscopy) effect have been successfully applied to study large biomolecular systems. In this perspective, we will discuss the fascinating early work with fluorinated aromatic amino acids, which reveals the enormous potential of these non-natural amino acids in biological NMR and the potential of 19F-NMR to characterize protein and nucleic acid structure, function and dynamics in the light of recent developments. Finally, we explore how fluorine NMR might be exploited to implement small molecule or fragment screens that resemble physiological conditions and discuss the opportunity to follow the fate of small molecules in living cells.

Covalently circularized nanodiscs for studying membrane proteins and viral entry.

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a ß-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.