Binding Location Research Articles

Cannabinoid non-cannabidiol site modulation of TRPV2 structure and function

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.

Computational Investigation of 5.5'',7''-trihydroxy-3,7-dimethoxy-4'-4''-O-biflavone from Flavonoids Using DFT Calculations and Molecular Docking

The structural characterization of the 5.5",7"-trihydroxy-3,7-dimethoxy-4'-4"-O-biflavone (TDOB) molecule was done in this study. Based on the molecule's stable phase geometry, entire calculations were done by use of the detailed CAM-B3LYP and PBEPBE approach with SDD and LanL2DZ. In addition, a variety of HOMO-LUMO energy ranges, natural bond inter-orbital and inter-orbital interactions, and electro-static surface mapping operations were as well carried out. The specific binding location and mechanism of the ligand on the protein were studied using molecular docking. In the study, affinity scores for TDOB-AKR1B1 (Aldose Reductase) (PDB: 4ICC) and (PDB: 4IGS) were found to be -8.559 cal/mol and -5.461 cal/mol, respectively. 4ICC receptor binding score was found to be greater. The inhibitory properties of TDOB were investigated against the enzymes 4ICC and 4IGS, both of which showed effective inhibition.

Insight into the binding behavior, structure, and thermal stability properties of β-lactoglobulin/Amoxicillin complex in a neutral environment

Antibiotics are extensively used in agriculture and husbandry, thus threatening human health due to their direct exposure to dairy products. Therefore, it is essential to investigate whether milk proteins are unintended carriers of antibiotics. In this research, therefore, we studied the impact of Amoxicillin on the conformation and structural stability of whey protein β-lactoglobulin by using spectroscopic techniques combined with molecular docking and simulation approaches. Further, the thermal stability of the resultant complex was explored. Amoxicillin was observed to effectively quench β-lactoglobulin's intrinsic fluorescence, suggesting that Amoxicillin was successfully bound to the β-lactoglobulin and a stable complex between them was established. The thermodynamic parameters demonstrated that van der Waals forces and hydrogen bonds dominated the spontaneous bonding process. According to circular dichroism (CD) and Fourier transform infrared (FTIR) analyses, the conformational structure of the β-lactoglobulin was altered upon the complexation with Amoxicillin. Further, this interaction significantly enhanced the thermal stability of β-lactoglobulin compared to sole β-lactoglobulin solutions. Molecular docking theoretically showed the preferred binding locus of Amoxicillin within the β-lactoglobulin structure. Molecular dynamics (MD) simulation results further validated the stability of the β-lactoglobulin-Amoxicillin complex. The findings obtained here are helpful for elucidating the potential of β-lactoglobulin to bind and transport harmful substances into the body, thus opening up new avenues for food security.

Characterization of Climbazole–Bovine serum albumin interaction by experimental and in silico approaches

Interactive association of an antifungal drug, climbazole (CBZ) with the carrier protein in bovine circulation, bovine serum albumin (BSA) was explored by fluorescence and absorption spectroscopy along with in silico techniques. The fluorescence and absorption spectral alterations of the protein upon addition of CBZ affirmed the complex foration between CBZ and BSA. The inverse temperature dependence behaviour of the KSV values as well as the hyperchromic result of the protein’s absorption signals characterized CBZ-triggered quenching of BSA fluorescence as the static quenching. A weak binding affinity (Ka = 3.12–1.90-× 103 M−1) was reported towards the CBZ−BSA association process. Interpretation of thermodynamic data (entropy change = +14.68 J mol−1 K−1 and enthalpy change = –15.07 kJ mol−1) and in silico analyses anticipated that hydrophobic forces, van der Waals forces and hydrogen bonds were the key intermolecular forces in the complex stabilization. Inclusion of CBZ to BSA produced microenvironmental perturbations around Tyr and Trp residues, and also significantly defended temperature-induced destabilization of BSA. The binding locus of CBZ was detected in the proximity of Sudlow’s sites I (subdomain IIA) and II (subdomain IIIA) of BSA, exhibiting greater preference towards site II, as revealed by competitive site-marker displacement investigations and in silico analysis. The stability of the CBZ–BSA complex was further validated by the molecular dynamics simulation assessments.

A novel insight into the binding behavior between soy protein and homologous ketones: Perspective from steric effect

The binding behavior between soy protein isolate (SPI) and homologous ketones was examined in this study. Three ketones (2-hexanone, 2-heptanone, and 2-octanone) with different carbon chain lengths were selected to investigate the interaction mechanism between SPI and ketones, and three ketones with positional isomers, namely 2-nonanone, 3-nonanone, and 4-nonanone, were used to construct different steric effect. Ultraviolet, fluorescence, and circular dichroism results showed that ketones induced the conformational perturbations of SPI, weakening the polarity of the environment in which the chromogenic group was located and triggering the quenching of SPI intrinsic fluorescence. Hydrophobic site competition experiments revealed that the presence of steric hindrance hindered the collision of compounds with SPI active site. The optimal binding locus for ketones within the SPI structure was determined by molecular docking, and two key amino acids, LYS412 and ASN484, were found. The calculated binding energy (-25.682 ∼ -17.952 kcal mol−1) and steric energy (7.883 ∼ 14.894 kcal mol−1) of ketones showed that steric effects played a negative role in the binding of SPI to flavor compounds, but this role was weaker than hydrophobic interactions and hydrogen bonding. The improved understanding of the role of carbon chain lengths and steric hindrance on ketone retention could support the flavor product designer or scientists to optimize flavor retention and delivery.

Efficiency of C = Se as hydrogen bond acceptor in controlling regioselective amination of 5,7-dinitroquinazoline-4-selenone: Quantum mechanical, AIM and docking analysis by density functional method

Herein, we explored the theoretical aspect of reaction between 5,7-dinitroquinazoline-4-selenone and methylamine by employing density functional theory (DFT) calculations. The energetic parameters confirmed that the reaction progressed via. concerted mechanism with the single step process instead of two step process via intermediate formation. Further, role of Se = C bond as non-conventional H-bond acceptor is reported in reaction controlling factors that monitors the regioselectivity of the present proposed reaction. Despite of substantial steric hindrance at ortho-position of 5,7-dinitroquinazoline-4-selenones, the regioselective amination resulted into ortho-substituted product (P2) rather than para-substituted product (P1) attributed to non-conventional NH---Se = C intramolecular hydrogen bonding. The topological properties of the electron density distributions have been studied by using quantum theory of atoms in molecules (QTAIM). The presence of non-conventional hydrogen bond acceptor–donor interactions (NH---Se = C) in ortho-positioned amination is achieved by electron density ρ(r) and Laplacian of electron density ∇2ρ(r) in QTAIM analysis and supported the results of DFT studies hybrid with B3LYP/6-31G(d,p) basis set. Due to the lack of intramolecular hydrogen bonding interactions (NH---Se = C), the para-positioned amination on 5,7-dinitroquinazoline-4-selenone is founded to be less feasible. Further, the DFT technique has been used to investigate frontier molecular orbital (FMO) analysis and global reactive parameters using the aforesaid level of theory and basis set. HOMO-LUMO analysis revealed that ortho-substituted product (P2) has a wider energy gap (3.0684 eV) than para-substituted product (P1) (2.6402 eV) suggesting more stability of P2. The presence of hydrogen bonding interactions is further confirmed by the change in electrostatic potential regions in ortho-substituted product (P2) as compared to reactants in molecular electrostatic potential surface (MEPS) studies. Both para-substituted product (P1) and ortho-substituted product (P2) have been docked into the protein 5FCT's active site to predict favored binding location, activity and affinity by using docking studies.

Novel Small Molecule and Peptide Inhibitors of CREB in Leukemia Cells

Background: Acute Lymphoblastic Leukemia (ALL) and Acute Myelogenous Leukemia (AML) account for nearly 45% of all childhood cancer. Current therapy for acute leukemia results in significant morbidity and late complications in children. Therefore, more effective and less toxic therapies to treat leukemia are needed. Previous work in our laboratory has identified CREB (cAMP Response Element Binding Protein), a transcription factor that promotes cell proliferation and survival in acute leukemia, is overexpressed in primary ALL and AML cells and is a potential target for therapy. We have investigated small molecules and peptides to inhibit the critical interaction between the phosphorylated KID (Kinase-Inducible) domain of CREB and the KIX (KID-interacting) domain of the coactivator CBP (CREB Binding Protein). Methods: We assessed a library of previously untested alpha-mimetic compounds from ChemDiv designed to mimic interactions between α-helices and protein surfaces for predicted interaction with a shallow hydrophobic groove on the interaction surface of KIX. We interrogated the 24,737 compounds in the library using rigid-receptor ligand docking, followed by induced-fit ligand docking of the top compounds using two different software, MOE and Schrodinger's Bioluminate. The top 50 binding poses from each software's rankings were combined to yield 87 candidate alpha-mimetic molecule inhibitors. In addition to our screen of small molecules, we have designed peptide inhibitors that closely mimic the αB helix of pKID. We investigated two methods to enhance the stability and cell-penetrating ability of our peptides: hydrocarbon stapling and the utilization of a rigid scaffold from the scorpion toxin peptide BmBKTtx1. Based on the calculated binding scores and locations, the most promising chimeric peptide designs were further investigated for binding stability using 500 nanosecond molecular dynamics (MD) simulations modeling their interaction with KIX. Results: The binding poses of the final set of small molecules were visually inspected for occupation of key hotspots on the pKID:KIX interaction surface, including the location of the phosphorylation sight of KID. Of the 87 molecules and their binding poses, only 21 of them (24%) obstructed fewer than two of the three hotspots chosen for the analysis. Moreover, 80 of the molecules (92%) extended into the hydrophobic pocket, the most critical hotspot. The multi-residue construction of this pocket likely contributed to the high similarity in interaction fingerprint between these molecules and pKID; occupation of this pocket can serve as a good indicator of strong ligand binding to KIX in future assays. The most-promising peptide designs using the scorpion toxin scaffold were assessed with MD simulations and compared to a control peptide comprised primarily of the αB helix of pKID. The chimeric peptides showed increased binding stability over the control peptide with an average RMSD of 6.8Å vs. 12.5Å, illustrating the benefits of the stability afforded by the scorpion scaffold design. Several of the hydrocarbon stapled peptides were tested in vitro using fluorescence polarization (FP) assay developed by our lab (Chae et al. Bioorganic & Medicinal Chemistry Letters, 2019) and in AML HL-60 and KG-1 cells and were shown to have growth inhibitory effects by Cell-Titer Glo Assays. Discussion: These small molecules and scorpion toxin-derived peptides represent novel candidate inhibitors of the pKID:KIX interaction implicated in leukemia. Based on promising results from our in silico analyses of these inhibitors, we have obtained ten structurally diverse alpha-mimetic small molecules and three chimeric peptides and aim to test their efficacy in vitro using the FP assay. Furthermore, we will obtain high resolution cryo-EM structures of successful compounds and peptides bound to the KIX domain using a double-shell protein scaffold (Zhang et al. ACS Central Science, 2022), and use these structures for further refinement of the inhibitors. The development of new inhibitors for the pKID:KIX interaction promises to reveal insights into the structural relationship between the two domains and the basis for effective, targeted leukemia therapeutics.

37. Epigenetic reprogramming of brain development pathways during non-small cell lung cancer metastasis to brain

Non-small cell lung cancer (NSCLC) is one of the most common and deadliest cancers, with roughly half of all patients initially presenting with both primary and metastatic disease. While the major events in the metastatic cascade have been identified, a mechanistic understanding of how NSCLC routinely, successfully colonizes the brain is largely unknown. To better understand this process, we profiled a combination of genomic, transcriptomic, and methylomic landscapes of 45 paired NSCLC primary and brain metastasis samples. 75 genes displayed recurrent metastasis enriched variants, largely implicated in focal adhesion and extracellular matrix receptor interactions. Variant allele frequencies over a wide range of epigenetic regulators displayed an increase in metastases, suggesting that epigenetic misregulation may be selected for and possibly contribute to NSCLC metastasis to brain. Consistent with these observations, we observed widespread changes in DNA methylation throughout disease progression, many found within brain-specific active enhancers and correlated with increased nearby gene expression. The greatest recurrent methylation changes during metastatic progression occurred over a subset of DNA methylation valleys (DMVs) enriched for H3K9me3 and bivalent marks H3K27me3 and H3K4me1 in normal lung. Mapping EZH2, the catalytic subunit of polycomb repressive complex 2 (PCR2), binding locations in H1299, a lymph node-derived lung cancer cell line, revealed a pervasive loss of EZH2 binding within DMVs accompanied by an increase in DNA methylation, exemplifying epigenetic switching. The vast majority of these DMR-associated DMVs harbor developmental genes, suggesting that altered epigenetic regulation of developmentally important genes may confer a selective advantage during metastatic progression.

Anionic lipids unlock the gates of select ion channels in the pacemaker family.

Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.

Characterization of a novel natural tetracenomycin reveals crucial role of 4-hydroxy group in ribosome binding

The aromatic polyketides tetracenomycins were recently found to be potent inhibitors of protein synthesis. Their binding site is located in a unique locus within the tunnel of the large ribosomal subunit. Here we report the isolation and structure elucidation of a novel natural tetracenomycin congener – O4–Me-tetracenomycin C (O4–Me-TcmC). This compound is isomeric to tetracenomycin X (TcmX), however, in contrast to TcmX, O4–Me-TcmC exhibited no antimicrobial activity and was unable to inhibit protein synthesis in vitro. Structural alignment of tetracenomycins to the binding locus from cryo-EM TcmX-70S ribosome data revealed the crucial role of the 4-hydroxyl group. These findings are important for further development of semi-synthetic tetracenomycins as potential antibacterials.

Detection and analysis of phage M13KO7 using biosensor based on imaging ellipsometry

Antibody GP8 (anti-GP8) was used as a ligand to detect phage M13KO7 using a biosensor based on imaging ellipsometry (IE) without oriented ligand immobilization. When the phage M13KO7 in a sample interacted with the antibody in a microarray, a complex formed upon their affinity, and the layer covering the surface cell of the interaction became thicker. The variation in layer thickness was represented by gray-scale (or brightness) variation of the IE image. Direct and oriented immobilization of the antibody were analyzed to identify the best binding locations to capture the phage. In direct immobilization, the phage could be captured by anti-GP8 and not antibody GP3 (anti-GP3), which was attributed to different copy numbers of the coat proteins on the body of the phage specific to the two antibodies. This indicated that different binding locations could produce different detection efficiency. In oriented immobilization, the phage could be captured by the protein G‒Fc-anti-GP3 model, with a low detection efficiency. In contrast, the avidin‒biotin-based oriented immobilization could capture the phage with a high detection efficiency. Specifically, it could expose more Fab domains of anti-GP3 labeled with biotin; one avidin had four biotin-binding sites. To confirm detection and analyze binding location, the phage M13KO7 was imaged by transmission electron microscopy and atomic force microscopy. Our results suggest that binding location plays a significant role in the detection of big targets and contributes to enhancing biosensor detection sensitivity.

The protocol of CUT&Tag for metabolic tissue cells.

Cleavage under target and tagment (CUT&Tag) is a technology that utilizes the fusion protein of Tn5 transposase and protein A/G which can guide Tn5 enzyme to the antibody bound to target protein and cleave the chromatin regions adjacent to target protein. Chromatin libraries are then tagged and sequenced by the high-throughput sequencing to obtain chromatin information at specific sites or protein binding locations. CUT&Tag technology plays an important role in the research of DNA and protein interactions. It can be used to understand the modifications of histone and the bindings of transcription factors. Compared with the traditional chromatin immunoprecipitation-sequencing (ChIP-seq) technology, the CUT&Tag has the strengths of high signal-to-noise ratio, good repeatability, short experimental period, and low cell input. It shows great advantages in early embryonic development, stem cells, cancer, epigenetics and other research fields. In this article, we described the protocol of CUT&Tag for metabolic tissue cells (mouse primary islet cells), to provide an epigenetic method for studying metabolic cells.

Adsorbate chemical environment-based machine learning framework for heterogeneous catalysis

Heterogeneous catalytic reactions are influenced by a subtle interplay of atomic-scale factors, ranging from the catalysts’ local morphology to the presence of high adsorbate coverages. Describing such phenomena via computational models requires generation and analysis of a large space of atomic configurations. To address this challenge, we present Adsorbate Chemical Environment-based Graph Convolution Neural Network (ACE-GCN), a screening workflow that accounts for atomistic configurations comprising diverse adsorbates, binding locations, coordination environments, and substrate morphologies. Using this workflow, we develop catalyst surface models for two illustrative systems: (i) NO adsorbed on a Pt3Sn(111) alloy surface, of interest for nitrate electroreduction processes, where high adsorbate coverages combined with low symmetry of the alloy substrate produce a large configurational space, and (ii) OH* adsorbed on a stepped Pt(221) facet, of relevance to the Oxygen Reduction Reaction, where configurational complexity results from the presence of irregular crystal surfaces, high adsorbate coverages, and directionally-dependent adsorbate-adsorbate interactions. In both cases, the ACE-GCN model, trained on a fraction (~10%) of the total DFT-relaxed configurations, successfully describes trends in the relative stabilities of unrelaxed atomic configurations sampled from a large configurational space. This approach is expected to accelerate development of rigorous descriptions of catalyst surfaces under in-situ conditions.

Intermolecular Interactions in G Protein-Coupled Receptor Allosteric Sites at the Membrane Interface from Molecular Dynamics Simulations and Quantum Chemical Calculations.

Allosteric modulators are called promising candidates in G protein-coupled receptor (GPCR) drug development by displaying subtype selectivity and more specific receptor modulation. Among the allosteric sites known to date, cavities at the receptor–lipid interface represent an uncharacteristic binding location that raises many questions about the ligand interactions and stability, the binding site structure, and how all of these are affected by lipid molecules. In this work, we analyze interactions in the allosteric sites of the PAR2, C5aR1, and GCGR receptors in three lipid compositions using molecular dynamics simulations. In addition, we performed quantum chemical calculations involving the symmetry-adapted perturbation theory (SAPT) and the natural population analysis to quantify the strength of intermolecular interactions. We show that besides classical hydrogen bonds, weak polar interactions such as O–HC, O–Br, and long-range electrostatics with the backbone amides contribute to the stability of allosteric modulators at the receptor–lipid interface. The allosteric cavities are detectable in various membrane compositions. The availability of polar atoms for interactions in such cavities can be assessed by water molecules from simulations. Although ligand–lipid interactions are weak, lipid tails play a role in ligand binding pose stability and the size of allosteric cavities. We discuss physicochemical aspects of ligand binding at the receptor–lipid interface and suggest a compound library enriched by weak donor groups for ligand search in such sites.

SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope

Population antibody response is thought to be important in selection of virus variants. We report that SARS-CoV-2 infection elicits a population immune response that is mediated by a lineage of VH1-69 germline antibodies. A representative antibody R1-32 from this lineage was isolated. By cryo-EM, we show that it targets a semi-cryptic epitope in the spike receptor-binding domain. Binding to this non-ACE2 competing epitope results in spike destruction, thereby inhibiting virus entry. On the basis of epitope location, neutralization mechanism and analysis of antibody binding to spike variants, we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of the identified population antibody response. These substitutions, including L452R (present in the Delta variant), disrupt interactions mediated by the VH1-69-specific hydrophobic HCDR2 to impair antibody-antigen association, enabling variants to escape. The first Omicron variants were sensitive to antibody R1-32 but subvariants that harbour L452R quickly emerged and spread. Our results provide insights into how SARS-CoV-2 variants emerge and evade host immune responses.

The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1)

Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (ScATE1) in the apo form. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.

Computational Studies on Antibody Drug Conjugates (ADCs) for Precision Oncology

AbstractAfter decades of technological research, the basic understanding of Antibody‐drug conjugates (ADCs) has resulted in the development of therapeutic agents for cancer patients. In this work, we have studied the mechanism of only nine FDA‐approved ADCs (Nat Rev Clin Oncol. 2021;18(6):327‐344) by computational methods, while many more ADCs are in preclinical and clinical development. The biological and Absorption, distribution, metabolism, excretion, and toxicity (ADMET) risk prediction for cytotoxic payloads is estimated to predict their bioavailability as drugs for the treatment of cancer patients. Other potential targets for the cytotoxic payloads are accessed by SwissTargetPrediction. Docking for the optimized structures of drugs and linkers are carried out by AutoDock tools. CABS‐flex 2.0 web server is used for Molecular Dynamics (MD) simulations of antigens and antibodies (IgG1, IgG4) and potential binding pockets for antibodies are searched by the PrankWeb server. HDOCK web server is used to find the docking of (Antigens‐ Antibodies‐ (linker‐payloads)) complexes. Protein‐ligand interaction profiler (PLIP) web server is used to find the noncovalent interactions in ADCs. Results indicated higher toxicity for the studied payloads, yet drug likeliness is observed for all studied cytotoxic payloads. The predicted targets for the payloads are mostly phosphodiesterase and protease electrochemical transporter. Strong Hydrogen bond Interactions have been observed for the ADCs. The cytotoxic payloads showed a specific binding location for the target antigens. Hopefully, these studies will help to improve the design patterns and facilitate the optimal allocation of ADCs for precision oncology in the future.

Structure of an iminosugar complex of a glycoside hydrolase family 5 lichenase provides insights into the active site

TmCel5B is a lichenase belonging to glycoside hydrolase family 5 subfamily 36 (GH5_36). To gain insights into the active site of this subfamily which contains multifunctional endoglycanases, we determined the crystal structure of TmCel5B in complex with an iminosugar, 1-deoxynojiromycin (DNJ). DNJ is bound to the −1 subsite, making a network of non-covalent interactions with the acid/base residue Glu139, the nucleophile Glu259, and with other residues that are conserved across the GH5 family. The catalytic site displayed a Glu-Arg-Glu triad of the catalytic glutamates that is unique to the GH5_36 subfamily. Structural comparison of active sites of GH5_36 homologs revealed divergent residues and loop regions that are likely molecular determinants of homolog-specific properties. Furthermore, a comparative analysis of the binding modes of iminocyclitol complexes of GH5 homologs revealed the structural basis of their binding to GH5 glycosidases, in which the subsite binding location, the interactions of the ligand with specific conserved residues, and the electrostatic interactions of the catalytic glutamates with the ring nitrogen, are crucial.

Sustainability Evaluation of Seaweed Aquabusiness Management in Support of Sustainable Development Goal in Ekas Bay, East Lombok

Highlight Research Based on technological and economic dimensions, the sustainability of seaweed cultivation in Ekas Bay was less sustainable (index value lied between 26 and 50). The distance between seed clumps, drying places, seedling binding locations, and warehouses were the most sensitive attributes from technological dimension. Market status, market scale, and target market were the most sensitive attributes from economic dimension Abstract Indonesia is one of the major countries in the world that produces seaweed. West Nusa Tenggara has become one of the centers of seaweed producers in Indonesia because it has many bays that are used for seaweed cultivation activities. The problem of seaweed aquabusiness is the low productivity of the production unit managed by seaweed farmers. This study aimed to assess the effectiveness and production level of seaweed cultivation technology and the success of seaweed cultivation by seaweed farmers in Ekas Bay based on technological dimensions (17 attributes) and economics (14 attributes). The Rapsewaqua application is used to analyze the sustainability status of seaweed cultivation. Based on the results of the analysis, the index value was 50.08 for the technology dimension and 39.50 for the economic dimension. The result showed that the sustainability of seaweed in Ekas Bay was in the less sustainable category. Spacing between seed clumps, drying area, seedling binding location, and warehouse were the most sensitive attributes on the technological dimension. Market status, market scale, and target market were the most sensitive attributes in the economic dimension. These sensitive attributes need to be improved to support the sustainability of seaweed in Ekas Bay.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein–protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.