In the crystal of the title compound, the molecules are linked into chains by N—H⋯O hydrogen bonds. The chains are interlinked by short Cl⋯N contacts. 16H12Cl4N2O4, is generated by a crystallographic centre of symmetry. In the crystal, N—H⋯O hydrogen bonds link the molecules into [010] chains featuring R22(10) loops. The chains are cross-linked by short Cl⋯N contacts [3.224 (2) Å].The complete molecule of the title compound, C The asymmetric unit consists of half a molecule, which is completed by inversion symmetry centred in the middle of the central N—N bond  is 77.8 (1)°; the latter has a crystallographically imposed trans conformation, in a manner similar to 2-[5-methyl-2-(propan-2-yl)phenoxy]-N′-{2-[5-methyl-2-(propan-2-yl)phenoxy]acetyl}acetohydrazide acetyl]acetohydrazide monohydrate acetoyl]hydrazine dihydrate .The twist angle between the 2,4-dichlorophenoxy ring system and the c-axis direction. The contact involves the para Cl atom of the 2,4-dichlorophenoxy group and the nitrogen atom of the N′-acetylacetohydrazide group, with a Cl2⋯N1 distance of 3.224 (2) Å .In the crystal, each molecule is involved in four N—H⋯O hydrogen-bonding contacts, donating two and accepting two bonds, leading to the formation of ribbons propagating parallel to [010] Table 1. The ribI) in 67% yield; m.p. 249–250°C acetohydrazide  and ethyl 2-cyano-3-ethoxyacrylate  in anhydrous ethanol (10 ml) was heated under reflux for 2 h. The solid obtained on cooling was collected by filtration, washed with ethanol, dried, and recrystallized from dimethylformamide solution to give colourless plates of  I. DOI: 10.1107/S2414314621003187/hb4380Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621003187/hb4380Isup3.cmlSupporting information file. DOI: 2073258CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In-silico analysis shows that DTN possess stable binding with protein targets involved in gastric ulcer pathophysiology. DTN exhibited an inhibitory effect against 2,2-diphenyl-1-picrylhydrazyl, H. pylori and hydrogen potassium ATPase (H+/K+-ATPase). The antiulcer activity was performed using an ethanol-induced gastric ulcer model in rats. Anti-oxidant profile of DTN showed a significant increase in glutathione-S-transferase, glutathione and catalase levels whereas lipid peroxidation levels were reduced. Histopathological findings confirmed that DTN protected the gastric mucosa of rats. Inflammatory markers tumor necrosis factor-alpha, nuclear factor kappa B, cyclooxygenase-2, interleukin 6 and interleukin-1β were reduced and prostaglandin-E2 restored expression of these cytokines in DTN pretreated animals when analyzed by using immunohistochemistry, enzyme-linked immunosorbent assay and western blot techniques. In real-time polymerase chain reaction technique, the expression of H+/K+-ATPase was downregulated in DTN pretreated group. DTN did not cause any mortality up to 400 mg/Kg. This study indicates that the newly synthesized compound DTN, possess stable binding against selected targets. DTN exhibits a gastro-protective effect, mediated via anti-H. pylori, H+/K+-ATPase inhibition, anti-oxidant and anti-inflammatory pathways, exploring its therapeutic potential in gastric ulcer management.The present study aims to investigate the newly synthesized organotin (IV) complex  dibutylstannanediyl bis (4-(4-nitrophenyl) amino)-4-oxobut-2-enoate (DTN) for its anti-ulcer potential. Characterization performed by carbon nuclear magnetic resonance spectroscopy proved that all values are in the expected ranges of the new compound. Gastroprotective activity of DTN was evaluated through The online version contains supplementary material available at 10.1186/s40360-022-00596-0. H. pylori bacteria is considered one of the major causes of gastric ulcers in humans [+/K+-ATPase pump where K+ pumps in and Na+ out and suppression of blood flow in gastric mucosa leads to gastric ulceration [Gastric ulcer is one of the most common chronic gastrointestinal diseases characterized by a significant defect in the mucosal barrier. About 5 to 10% of people were infected and a major public health burden in last two decades . H. pylon humans . Hyperseceration . Oxidaticeration . On the ceration .The ethanol-induced gastric ulcer model resembles gastric ulcer disease in humans , 8. Gastn = 1–4. Depending on the number of organic moieties (n) they are connected to the Sn atom the organotin (IV) compounds are classified as RSnX3, R2SnX2, R3SnX and R4Sn: and are called mono, di, tri and tetra organotin (IV) compounds [Organometallic compounds containing Sn-C atoms are linked to the organic moiety direct Sn-C covalent bonds are known to explode with organotin (IV) compounds. The general formula of (RnSnX4-n) where R contains organic matter, X, any anionic group such as like Cl−, OH– etc. and ompounds . Tin metin-silico, in-vitro, in-vivo and ex-vivo proteomic analysis techniques.Organotin (IV) compounds have many applications in non-biological and biological aspects . Organot2 ELISA kit  were purchased from Elabscience, China. All chemicals were used in experiments analytically approved (99% HPLC grade).Dibutyltindichloride, maellicanhydride, 4-Nitro aniline, ethanol, dimethyl sulfoxide (DMSO), chloroform and normal saline acquired from Sigma-Aldrich, Germany. DTN compound was prepared in postgraduate chemistry research laboratory of Riphah Institute of Pharmaceutical Sciences (RIPS) Islamabad, Pakistan. Omeprazole, metronidazole purchased from Barrett Hodgson and Sanofi Aventis. Secondary antibodies procured from Abcam UK. Rat NF-ĸB ELISA kit , rat TNF-α ELISA kit , rat IL-6  and rat IL-1β  and rat PGESprague-Dawley rats (180-240 g) of both sex have been used in the experimental process kept in animal house of the Riphah Institute of Pharmaceutical Sciences (RIPS) Islamabad, Pakistan, with organized environment provided (20-25 °C). Experiment trials were performed according to the rules and regulations of Research and Ethics Committee RIPS (Ref. No. REC/RIPS/2021/018) along with the guidelines of “Principles of Laboratory Animal care “.3) and stirred continuously for 15-20 minutes. In the next step, NaL was reacted with equimolar quantities of dibutyltindichloride in dried toluene solvent and refluxing for 8–12 hrs at temperature 110 °C. TLC was employed to check the reaction progress, after completion reaction mixture was filtered, evaporated in vacuo to get final desired product DTN  (Bruker-AM 300spectrophotometer) was employed to determine the composition analysis of new compound [Sodium salt of ligand was (NaL) product was prepared by reacting the synthesized carboxylic acid with aqueous solution of . The three-dimensional structure (3D) of the newly synthesized test compound DTN was drawn in chem sketch. The 3D structure of standard drugs i.e. omeprazole, phenoxybenzamine, ranitidine, aspirin, meclofenamate, dinoprostone and curcumin was obtained from PubChem. Human protein targets involved in the pathogenesis of gastric ulcer were selected and 3D structures were acquired from the online protein database, Research collaboratory for structural bioinformatics (RCSB) PDB. The selected target proteins were histamine receptor H2 (PDB ID: P25021), hydrogen potassium ATPase H+/K+-ATPase (PDB ID: 5ylu), muscarinic receptor M1 (PDB ID: 5CXV), prostaglandin-E2 PGE2 (PDB ID: 6AK3), Tumor necrosis factor-alpha TNF-α (PDB ID: 1BKC), nuclear factor kappa B NFĸB (PDB ID: 4Q3J), cycloxygenase-1 COX1 (PDB ID: 6Y3C) and cycloxygenase-2 COX2 (PDB ID: 5IKQ). Ligand water molecules were removed in discovery studio visualizer (DSV) and H (polar) atoms were added and saved in PDB format. Auto dock tools 1.5.6 and Auto Dock 4.2 docking software were used for molecular docking. The results were analyzed as the atomic contact energy  . One best pose with lowest ACE value  was selected for post-dock analysis via Biovia DSV. By determination of the interactions between ligand and amino acid residues were evaluated through 2D images [Docking studies were performed in Autodock 4.2 and CDOCKER. The parameter file of Autodock was modified due to presence of metal in ligand structure thus needed parameters which were obtained from the Autodock website  and the interaction energy, which indicate ligand binding affinity are calculated. The crystal water molecules are generally removed in rigid and semi-flexible docking process , since t2,2-diphenyl-1-picrylhydrazyl (DPPH) stock solution was prepared in 100 mL methanol by dissolving 9.2 mg DPPH. The ascorbic acid stock solution was prepared by dissolving (1 mg/mL) DMSO. Four milligrams compound was dissolved in DMSO for the preparation of the stock solution. These samples were tested for their potential of scavenging free radicals. In each well of 96 well microplates, test samples were transferred to the relevant well and then (190 μL) DPPH reagents were added to each well. 200 μg/mL was the final sample concentration. The reaction mixture was incubated in the dark at 37 °C; the absorbance was measured at 517 nm . Free raWhere, Abs (sample absorbance), Abc (Absorbance of negative control). Triple serial dilution were  concentrations.Helicobacter pylori (H. pylori) was evaluated through disc diffusion method [H. pylori j63 (cagA−), j196 (cagA−) and j107 (cagA+) were obtained from the biopsy of the gastric ulcer patient, voluntarily at care endoscopy clinics and labs . Biopsies were placed in modified thio campy medium. The plates were incubated at 37 °C under microaerophilic environment. The isolates were identified by morphology and urease test kit. The isolates were kept in sterile McCartney bottles containing 0.2 g / L of cysteine and 20% of glycerol in brain heart infusion BHI broth 80 °C. Frozen clinical isolates were inoculated on Muller-Hinton Agar (MHA) plates. DTN test compound with different concentrations were added onto standard discs and was placed on the MHA plate. After incubation at 37 °C for 3 to 5 days, the zone inhibition was measured for each disk. All tests were performed in triplicate and antibacterial activity was evaluated as the mean of the inhibition diameter (mm). Metronidazole was used as a positive control in the experiment [The antibacterial activity of DTN compound against n method . Three dperiment .n = 5). Group (I) served as a saline control received saline solution of (10 mL/Kg) body weight. Group (II), (III) and (IV) pretreated with DTN at doses of 5, 10 and 30 mg/Kg (p.o) respectively and group (V) received omeprazole (20 mg/Kg) served as standard drug and group (VI) was negative control received absolute ethanol. One hour after all treatments, absolute ethanol (1 mL/100 g) was administered orally to each rat. After 1 hour of ethanol treatment all rats were euthanized by cervical displacement and the stomachs were removed and saline normal solution was used to washed them and the lesion index was estimated by measuring each lesion in mm along its largest curvature scored area of each lesion as measured and marked according to the method previously described by [For induction of gastric lesions, after 24 hours of fasting rats were randomly assigned to six different groups . Gastric-tissues kept in biofreezer (− 80 °C) were homogenized using Silent Crusher M (heidolp). The homogenate was then centrifuged for 10 minutes at 3500 rpm and the supernatant was separated. Supernatant was analyzed for release of inorganic phosphate after ATP hydrolysis spectrophotometrically at 660 nm. One ATPase activity unit has been described as one micro mole of inorganic phosphorus released by ATP hydrolysis through ATPase 1 mg per hour tissue protein. It was then expressed as μmol pi/mg prot/hour [DTN inhibitory effect on rat gastric Hrot/hour .Gastric tissue isolated from the rat was homogenized and then centrifuged at 1500 rpm for 30 min. and the supernatant was collected. Supernatant was then estimated for glutathione (GSH), catalase, glutathione-S-transferase (GST) and lipid peroxidation (LPO) levels. GSH levels were determined by oxidation of GSH and DTNP which gave a yellow end product. The absorbance of 2-nitro-5-thiobenzoic acid was calculated at 412 nm with the help of a GSH microplate reader. Values expressed in μmoles/mg of proteins GST activity calculated by extinction coefficient of the product formed and expressed in μmole/mg of CDNB conjugate/min/mg of protein. The GST level was determined by the formation of CDNB conjugate and measured its absorbance at 340 nm. Degradation of H2O2 measured in the presence of catalase at 240 nm absorbance was measured using a catalase microplate reader. The LPO level was analyzed by its resultant end product called malondialdehyde (MDA). Microplate reader was used to measure the absorbance at wavelength of 532 nm where quantitative measurement of LPO was expressed in TBARS nmoles/min/mg of protein .Tissue sections were deparaffinized with absolute xylene (100%) on coated slides and then rehydrated with ethyl alcohol (from 100% (absolute) to 70%). The slides were cleaned with distilled water and immersed for 10 min in hematoxylin. The slides were then placed under running water for 10 minutes in glass jar and treated with 1% HCl and 1% ammonia water. The slides were added for 5–10 min to eosin solution. After due time, the slides were washed in water and they are air dried for some time. The dried slides were dehydrated in graded ethyl alcohol . The slides were cleaned with xylene and were mounted with a glass cover slip. Images of slides were taken using a light microscope  and analyzed through Image J, a computer-based program, while focusing thoroughly on gastric cell size and shape, inflamed infiltrated cells, and vacuolation. The TIF images were arranged to the same threshold intensity for all groups and examined in the GraphPad Prism.8 .Immunohistochemical analysis was performed as described previously by . After d2), TNF-α, interleukin 6 (IL-6) and interleukin 1 beta (IL-1β) was performed following the manufacturer’s (Elabscience) instructions. Stomach tissues were homogenized using Silent crusher-M (Heidolph) at 15 rpm × 1000. The supernatant was collected after centrifugation at (1350 X rpm for 15 minutes). With the BCA method, the total protein concentration in each group was determined. In brief, the protein samples were treated with the corresponding antibodies provided by kit. By using an ELISA microplate reader, the concentration of p-NFĸB  prostaglandins E2 (PGE2) , and TNF-α , IL-6  and IL-1  determined. All measures were taken in triplicates [ELISA of p-NFĸB, prostaglandins E2  protein assay kit protein concentration was measured. The 30 μg protein homogenate was fixed onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shifted to a polyvinylidene fluoride membrane. Membranes were incubated with primary antibodies, such as p-NFκB and TNF-α, overnight at 4 °C then blocked with 5% bovine serum albumin for 1 h at room temperature. After cleaning three times using tris-buffered saline with 0.1% Tween 20, the membranes proceeded with a 1:1000 dilution of secondary antibodies, such as goat, anti-rabbit for 90 min at room temperature. To visualize the immuno-reactive bands, an enhanced western blotting substrate kit was used. Using Image J software, densitometry evaluated the quantification of protein expression .-∆∆CT method with slight modification [+/K+-ATPase were CCCGCGAGTACAACCTTCT (forward) and CGTCATCCATGGCGAACT (reverse) and TATGAATTGTACTCAGTGGA (forward) and TGGTCTGGTACTTCTGCT (reverse), respectively.According to the manufacturer’s instructions, total ribonucleic acid (RNA) was extracted from the gastric tissues, by using the Trizol method. The first-strand of cDNA was generated from 1 to 2 μg of total RNA by a reverse transcriptase enzyme mix on a PCR thermocycler. The mRNA expression normalized to the expression of β-Actin as a house-keeping gene by 2fication . Primer Toxicity studies of DTN was performed, which defines the lethal dose versus the non-lethal dose of the test compound in the animal model. Using the acute toxicity model rats were divided into two groups of five rats each. Control group treated with normal saline (10 mL/Kg). By using increasing doses of test compound, the test was performed DTN (50–400 mg/Kg) administered orally in normal saline (10 mL/Kg). Forty-eight hours after treatment the rats were observed for mortality .P < 0.05.Results data were calculated with mean ± standard error mean of sample (mean ± SEM)). Image J software of (NIH) was used for morphological data analysis. Results were computed by using of one-way analysis of variance (ANOVA) with applied post hoc turkey’s test by using software (Graph pad prism version 8). Level of significance was considered 28H32N4O10Sn and 13C NMR confirmed the structure of synthesized compound as 14.0 (CH3), 26.1(CH2 butyl), 125.4(C=C), 142.6(C=C), 112.8, 119.3, 126.8, 131.4, 136.0, 145.8(Aryl C), 156.1(C=O), 164.8(C=O). Butyl carbons were observed upfield at 14.0-26.1 ppm. Alkenyl carbon atoms were observed downfield at 125.4 and 142.6 ppm respectively, the most downfield signals were attributed to carbonyl groups of ester and amide moieties. All the aryl carbons resonated in the expected ranges.A newly synthesized compound of organotin (IV), chemical formula is C+/K+-ATPase pump, muscarinic receptor M1, histamine receptor H2, COX1, COX2, PGE2, NFĸB and TNF-α using AD and CD (ACE and H-bonding only). Figures  of − 9.12 and − 9.88  and formed 4 and 1 H-bonds and 5 hydrophobic interactions and standard drug omeprazole showed  -8.2 and − 11.63  formed 2 and zero H-bonds and 6 hydrophobic interactions respectively. Using AD and CD, DTN showed an  -9.44 and − 16.39  against M1 receptor and formed 5 and zero H-bonds and 6 hydrophobic interaction and standard drug phenoxybenzamine showed  -8.5 and − 13.38  formed 1 π-π bond and 5 hydrophobic interactions. By employing AD and CD, DTN showed  against the H2 receptor formed − 7.10 and − 10.37  and formed 2 and 1 H-bonds and 5 hydrophobic interactions and standard drug ranitidine showed  -6.9 and − 15  formed 1 and zero H-bond and 5 hydrophobic interactions respectively. Using AD and CD against COX1, DTN showed  -6.46 and − 6.95  and formed 2 and 2 H-bonds and 5 hydrophobic interaction and standard drug aspirin showed  -6.2 and − 6.61  formed 3 and zero H-bonds and 2 hydrophobic interactions. Against COX2 target by using AD and CD, DTN showed an  of − 10.64 and − 13.89  and formed 3 and 2 H-bonds and 1 π-π bond and 3 hydrophobic interactions and standard drug meclofinamate showed  -8.42 and − 8.94  formed 4 and 2 H-bonds and 5 hydrophobic interactions. Against PGE2 target by employing AD and CD, the DTN represents  -8.98 and − 18.63  and formed the 2 and 2 H-bonds, 1 π-π bond and 5 hydrophobic interactions and standard drug dinopristone showed  -8.24 and − 14.82  formed 5 and 1 H-bonds and 1 hydrophobic interaction. Against NFĸB target through AD and CD, DTN showed an  -4.03 and − 5.64  and formed 3 and 2 H-bonds 1 π-π bond and 3 hydrophobic interactions and standard drug curcumin showed  -7.13 and − 8.54  formed 4 and 1 H-bonds and 3 hydrophobic interactions. Using AD and CD against TNF-α receptor, DTN showed  -8.06 and − 15.6  and formed 5 and 1 H-bonds and 5 hydrophobic interactions and standard drug aspirin showed  -5.20 and − 6.13  formed 4 and zero H-bonds and 1 hydrophobic interaction respectively.DTN compound showed significant binding to various protein receptors when evaluated using AD and CD. Table Figures SA represe50 effect at dose concentration on (7.4 μg/mL) and ascorbic acid showed IC50 at dose (4.3 μg/mL) respectively  showed compound anti-oxidant effect 70.13 ± 0.57, 60.27 ± 1.15, 57.83 ± 0.28, 44.32 ± 0.18 and 42 ± 0.46 and ascorbic acid at dose (20 μg/mL) showed 88 ± 0.55 anti-oxidant effect. DTN showed ICH. pylori activity against three different strains j63 (cagA-), j196 (cagA-) and j107 (cagA+) were determined by disc diffusion method, metronidazole was used as a positive control. Against strain-I, strain-II and strain-III different concentrations used of DTN and metronidazole were 0.5, 1, 2, 4, 8, 16, and 32 μg/disk and Inhibition diameter (mm) of DTN against strain-I was 2.33 ± 0.66, 3.66 ± 0.33, 4.66 ± 0.33, 6.0 ± 0.57, 10 ± 1.15, 12.66 ± 0.33, 15.33 ± 1 and metronidazole was 3.66 ± 0.33, 4.66 ± 0.33, 5.33 ± 0.66, 7 ± 0.57, 10.33 ± 1.20, 14.66 ± 0.88, 22 ± 1.15. DTN against strain-II was 3 ± 0.57, 3.66 ± 0.66, 5 ± 0.57, 6 ± 1.73, 9.33 ± 0.66, 12.66 ± 0.88, 19 ± 0.57 and metronidazole was 4 ± 0.57, 5 ± 0.57, 5 ± 0.57, 7.33 ± 0.88, 10.33 ± 1.20, 15 ± 1.73, 0.66 ± 2.30. DTN against strain-III was 2.66 ± 0.33, 4 ± 0.57, 4 ± 0.57, 6.33 ± 0.66, 10.33 ± 1.20, 14 ± 0.57, 18.66 ± 0.88 and metronidazole was 4 ± 0.57, 4.66 ± 0.33, 5.66 ± 0.88, 8 ± 1.15, 11.33 ± 0.66, 15.66 ± 0.33, 22.66 ± 0.66 respectively. MIC (μg/ml) of DTN effect against H. pylori was 12, 10, 10 and Metronidazole was 4, 6 and 4 (μg/ml) respectively  exhibited an antiulcer effect. DTN shows 41, 58 and 90% protective effect at 5, 10 and 30 mg/kg doses with ulcer index scores 10 ± 0.25, 5.9 ± 0.29, 4.2 ± 0.37, 1 ± 0.27 respectively. Omeprazole (20 mg/Kg) exhibited 90% inhibition with ulcer index 1 ± 0.22 as compare to ethanol group respectively . In ethanol group (1 mL/100 g) the level was significantly increased to 103.8 ± 0.44 (μmol Pi/mg prot/hour) as compared to saline group. In treatment group DTN (30 mg/Kg) expression of H+/K+-ATPase is 25.96 ± 0.44 and omeprazole (20 mg/Kg) was 30.29 ± 0.44 significantly decreased as compared to ethanol group  H202 /min/mg, 66.2 ± 0.66 CDNB conjugate/min/mg, 47 ± 0.63 μmoles/min/mg and 40 ± 0.70 (Tbars-nM/min/mg) respectively. In ethanol group (1 mL/100 g), Catalase, GST, GSH and LPO levels were 10 ± 0.63 μmoles H202/min/mg, 17 ± 0.63 CDNB conjugate/min/mg, 13 ± 0.70 μmoles/min/mg and 128 ± 1.2 (Tbars-nM/min/mg). In DTN treated group (30 mg/Kg) stomach tissues catalase, GST, GSH and LPO levels were 22 ± 0.70 μmoles H202 /min/mg, 48 ± 0.70 CDNB conjugate/min/mg, 22 ± 0.44 μmoles/min/mg and 72 ± 0.70 Tbars-nM/min/mg respectively. In Omeprazole (20 mg/Kg) treated group stomach tissues Catalase, GST, GSH and LPO levels were 25 ± 0.63 μmoles H202 /min/mg, 61 ± 0.63 CDNB conjugate/min/mg, 38 ± 0.63 μmoles/min/mg and 48 ± 0.70 Tbars-nM/min/mg respectively , Catalase, GST, GSH and LPO levels were 70 ± 0.31 μmoles HSaline (10 mL/Kg) group revealed normal stomach tissues architecture without any pathological changes. Ethanol (1 mL/100 g) treated tissues exhibited severe gastric damaged with vacuolation and disruption of morphological cell boundaries. DTN (30 mg/Kg) and omeprazole (20 mg/Kg) treated gastric tissues revealed the regeneration and restoration of stomach cells with mild degeneration respectively Fig. .Fig. 6HiIHC of gastric tissues revealed that ethanol (1 mL/100 g) treated group markedly upregulated inflammatory markers COX-2, p-NFκB and TNF-α expression. Vacuolation, necrotic cells and disruption of morphological cell boundaries were found in disease group. DTN (30 mg/Kg) and omeprazole (20 mg/Kg) group’s downregulated COX-2, p-NFκB and TNF-α expression Fig. .Fig. 7Sl2 level was 730 ± 50 pg/mg significantly decreased as compare to saline group. In DTN treated group (30 mg/Kg) p-NFĸB, TNF-α, IL-6 and IL-1 β levels were 880 ± 3.64, 2455 ± 75, 615 ± 1.1 and 1175 ± 0.34 pg/mg significantly decreased and PGE2 level was 1400 ± 50 pg/mg significantly increased as compare ethanol group. In omeprazole treated group (20 mg/Kg) p-NFĸB, TNF-α, IL-6 and IL-1 β levels were 505 ± 3.5, 2215 ± 65, 515 ± 0.99 and 850 ± 0.66 pg/mg significantly decreased and PGE2, 1765 ± 85 pg/mg significantly increased as compare to ethanol group respectively , p-NF-ĸB, TNF-α, PGE2, IL-6 and IL-1 β levels were 330 ± 9.52, 1955 ± 55 and 1770 ± 60 pg/mg, 415 ± 1.1 and 650 ± 0.89 pg/mL respectively. In ethanol group (1 mL/100 g) p-NFĸB, TNF-α, IL-6 and IL-1 β levels were 3855 ± 3.70, 3931 ± 40,685 ± 0.77 and 1625 ± 0.85 significantly increased and PGEIn ethanol group (1 mL/100 g), TNF-α and p-NF-ĸB expression in the gastric mucosa region were increased. In treatment group DTN (30 mg/Kg) and omeprazole (20 mg/Kg) suppressed the elevated expression of TNF-α and p-NF-ĸB Fig. .Fig. 9A +/K+-ATPase in ethanol-induced gastric ulcer. In ethanol treated group expression of H+/K+-ATPase mRNA levels increased. DTN (30 mg/Kg) and omeprazole (20 mg/Kg) decreased H+/K+-ATPase mRNA levels , exploring its therapeutic potential in gastric ulcer management. Characterization of a newly synthesized compound was performed to confirm the final product using different confirmatory methods.13CNMR techniques were used for further confirmation of the final synthesized product organotin (IV) complex. Identification tests results proved that the final product was confirmed and the bands of functional group of the desired product lies in range [The current study confirmed that newly synthesized test compound DTN exhibits anti-ulcer effect  dose decreases the expression of p-NF-ĸB, TNF-α and COX2 compared to the ulcer group [IHC staining of inflammation causing cytokines of ethanol-induced gastric ulcer was further experimentally examined. It revealed over expression of p-NF-ĸB, TNF-α and COXer group , 38. PGEer group . In a prer group . In ethaer group . As a reEthanol induces the inflammatory markers that initiates macrophages produce vast volumes of pro-inflammatory cytokines like TNF-α, p-NF-ĸB, IL-6 and IL-1β which promote the accumulation of neutrophils in the inflammation site, leading to the breakdown and the destruction of the mucosal barrier and ROS activates p-NF-ĸB through IĸB phosphorylation , 39. The+/K+-ATPase pump inhibition pathway that m-RNA levels of H+/K+-ATPase was determined in ethanol group. While treatment with DTN and omeprazole significantly reduced the level of H+/K+-ATPase compared to the ethanol group [H.pylori, H+/K+-ATPase inhibition, anti-oxidant and anti-inflammatory pathways. Due to multi effective property, it may reduce polypharmacy, be economically cost-effective, decrease medication error, drug-drug interactions and be effective in ulcer disease management.Western blot findings provide evidence that DTN has an anti-inflammatory effect through reduced expression of TNF-α and p-NF-ĸB. Both proteins are involved in the recruitment of inflammatory mediators, in the previous study it was revealed that the inflammatory mediator of TNF-α and p-NF-ĸB downregulated in ethanol-induced ulcerated tissue of the stomach . The RT-ol group . It was H.pylori, H+/K+-ATPase inhibition, anti-oxidant and anti-inflammatory pathways demonstrate its therapeutic potential in management of gastric ulcer.This study reveals that the newly synthesized compound DTN, possesses binding energy values − 4.03 to − 10.64 Kcal/mol against selected targets. DTN exhibits an anti-ulcer effect, mediated via anti-Additional file 1: Figure S1. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and omeprazole with hydrogen potassium atipase pump (H+/K+-ATPase) evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S2. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and phenoxy benzamine with muscarinic receptor (M1) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S3. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4- nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and ranitidine with histamine receptor (H2) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S4. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4- nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and aspirin with cyclooxiginase-1 (COX1) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S5. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and meclofenamate with cyclooxiginase-2 (COX2) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S6. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and dinopristone with prostaglandin-E2 (PGE2) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S7. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4- nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and curcumin with nuclear factor kappa B (NFĸB) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S8. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and aspirin with tumor necrosis factor alpha (TNF- α) respectively, evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S1A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and Omeprazole with hydrogen potassium ATPase (H+/K+-ATPase) evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S2A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and Phenoxy-benzamine with Muscarinic receptor (M1) evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S3A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and Ranitidine with histaminergic receptor (H2), evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S4A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and Aspirin with cyclooxygenase-1 (COX1), evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S5A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and Meclofinamate with cyclooxygenase-2 (COX2) evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S6A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl) amino)-4-oxobut-2-enoate (DTN) and Dinopristone with prostaglandin-E2 (PGE2), evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S7A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl)amino)-4-oxobut-2-enoate (DTN) and Curcumin with Nuclear Factor kappa-B (NFκB) evaluated through Biovia Discovery Studio Visualizer (DSV) 2016. Figure S8A. (A) and (B) represents 2D-interactions of 2E,2′E) dibutylstannanediyl bis(4-(4-nitrophenyl) amino)-4-oxobut-2-enoate (DTN) and aspirin with Tumor necrosis factor alpha (TNF-α) evaluated through Biovia Discovery Studio Visualizer (DSV) 2016.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


The compound was synthesized by addition of phenylselenyl bromide to a cyanamide.The title compound, C 25H22N2O2Se, crystallizes in the space group P21/c with one molecule in the asymmetric unit. The compound was synthesized by the addition of phenylselenyl bromide to a cyanamide. The phenylselenyl portion and the cyano group, as well as the ketone functional group in the cyclohexa-2,5-dien-1-one portion of the structure, are disordered, with occupancy factors of 0.555 (14) and 0.445 (14).The title compound, C The C1—Se1A—C18 bond angle is 101.2 (4)° and the C1—Se1B—C18A bond angle is 98.2 (5)°.The compound contains one phenylselanyl group that is disordered over two conformations, with occupancies of 0.555 (14) and 0.445 (14). The central five-membered ring is almost planar, with a maximum deviation for the C13 atom of only 0.133 (2) Å. In the spiro region of the molecule, the dihedral angle between the central five-membered ring and the cyclohexa-2,5-dien-1-one unit is 88.22 (7)°. The dihedral angle between the central ring and the methoxyphenyl group is only 55.98 (10)°, while the angle with the major component of the disordered phenylselanyl group is 88.6 (2)°. The Se1et al., 2016The title compound was synthesized and crystallized following the procedure reported by our group  I, global. DOI: 10.1107/S2414314620000784/bv4028Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620000784/bv4028Isup3.cmlSupporting information file. DOI: 1979402CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title compound, the 5-chlorophenol ring and the imidazole ring are nearly coplanar, with a dihedral angle of 15.76 (9)°. The ethylphenyl ring and the two phenyl rings subtend at angles of 71.09 (7), 43.95 (5) and 36.53 (9)°, respectively, with the imidazole plane. 29H23ClN2O, the 5-chlorophenol ring and the imidazole ring are nearly coplanar, with a dihedral angle of 15.76 (9)° between them. The ethylphenyl ring and the two phenyl rings subtend angles of 71.09 (7), 43.95 (5) and 36.53 (9)°, respectively, with the imidazole plane. An intramolecular O—H⋯N hydrogen bond supports the molecular conformation, and an intermolecular C—H⋯O interaction, originating from an ortho-phenyl H atom, stabilizes the packing arrangement. In addition, a weak C—H⋯π interaction, also involving an ortho-phenyl H atom, is observed.In the title compound, C GC–MS : m/z: 450.95.The title compound was synthesized by the one-pot reaction of benzil (10 mmol), 4-ethylaniline (10 mmol) and 5-chloro-2-hydroxybenzaldehyde (10 mmol) with ammonium acetate (10 mmol) in a glacial acetic acid (20 ml) medium. The mixture was refluxed for 5 h at 343 K, the progress of the reaction being monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature and poured into 100 ml of ice-cold water. The resulting precipitate was filtered, dried and further purified by column chromatography (7:3 petroleum ether:ethyl acetate) and isolated in good yield (85%). The product was recrystallized from ethanol solution. IR (KBr)  global, I. DOI: 10.1107/S2414314619016900/zl4037Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314619016900/zl4037sup3.tifSupporting information file. DOI: 10.1107/S2414314619016900/zl4037sup4.pdfSupporting information file. DOI: 10.1107/S2414314619016900/zl4037sup5.pdfProton-NMR. DOI: Click here for additional data file.10.1107/S2414314619016900/zl4037Isup6.cmlSupporting information file. DOI: 1950453CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Caenorhabditis elegans cadherin and catenin shows that these proteins are key for embryonic morphogenesis, we know surprisingly little about their structure and attachment to the cytoskeleton. In contrast to mammalian α-catenin that functions as a dimer or monomer, the α-catenin ortholog from C. elegans, HMP1 for humpback, is a monomer. Our cryogenic electron microscopy (cryoEM) structure of HMP1/α-catenin reveals that the amino- and carboxy-terminal domains of HMP1/α-catenin are disordered and not in contact with the remaining HMP1/α-catenin middle domain. Since the carboxy-terminal HMP1/α-catenin domain is the F-actin-binding domain (FABD), this interdomain constellation suggests that HMP1/α-catenin is constitutively active, which we confirm biochemically. Our perhaps most surprising finding, given the high sequence similarity between the mammalian and nematode proteins, is our cryoEM structure of HMP1/α-catenin bound to F-actin. Unlike the structure of mammalian α-catenin bound to F-actin, binding to F-actin seems to allosterically convert a loop region of the HMP1/α-catenin FABD to extend an HMP1/α-catenin FABD α-helix. We use cryoEM and bundling assays to show for the first time how the FABD of HMP1/α-catenin bundles actin in the absence of force. Collectively, our data advance our understanding of α-catenin regulation of cell–cell contacts and additionally aid our understanding of the evolution of multicellularity in metazoans.The regulation of cell–cell junctions during epidermal morphogenesis ensures tissue integrity, a process regulated by α-catenin. This cytoskeletal protein connects the cadherin complex to filamentous actin at cell–cell junctions. The cadherin–catenin complex plays key roles in cell physiology, organism development, and disease. While mutagenesis of  Cell–cell contacts are essential in the maintenance of the architecture of tissues and the communication between cells. At the center of these cell–cell junctions is the cadherin receptor. Upon binding to calcium, the extracellular domain of one cadherin binds to that of a neighboring cell. In the cytosol, the transmembrane cadherin binds β-catenin, which binds the cytoskeletal protein α-catenin. The resulting cadherin-catenin complex is key to cell physiology, organism development, and disease , 6, 7, 8Caenorhabditis elegans, proteins equivalent in structure and function to mammalian cadherin, β-catenin, and α-catenin are HMR1, HMP2, and HMP1, respectively  . The amiDespite over 39% sequence identity and 60% similarity along the lengths of human α-catenin and HMP1/α-catenin polypeptide chains, HMP1/α-catenin is a monomer , while mKey known α-catenin structures are the complex between α-catenin and β-catenin , dimericC. elegans has an α-catenin homolog, named HMP1 for humpback. HMP1/α-catenin shares over 39% sequence identity throughout the human α-catenin polypeptide chain . While vertebrate α-catenins are monomeric or dimeric depending on their location and function, the roundworm HMP1/α-catenin does not dimerize  α-catenin as a control that, in comparison, shows traces of possible dimerization as analyzed by size exclusion chromatography (SEC) and multiangle light scattering .The nematode dimerize  . We used the zebrafish (D. reiro) and the slime mold (Dictyostelium discoideum) α-catenin as controls.Loss of functions mutants of HMP1/α-catenin cause circumferential F-actin bundles to detach from the plasma membrane. This process results in the folding of the dorsal epidermis and the Humpback phenotype . HMP1/α-Fig. 1g) analyses allowed F-actin bundling readouts compared with high-speed sedimentation  analyses. In the absence of actin binding proteins, F-actin remains in the supernatant under low-speed conditions while F-actin pellets upon high-speed centrifugation.Truncations of the FABD of α-catenin affect its binding to F-actin . To put E and 2A). Recent total internal reflection fluorescence microscopy showed that bundling by the mammalian α-catenin FABD was increased upon motor activation (B and C). However, we only observed F-actin bundling when we assayed with the FABD but not with full-length HMP1/α-catenin (E). Further, full-length monomeric human α-catenin did also not bundle F-actin while full-length dimeric human α-catenin bundled F-actin (F). However, when truncating the carboxy terminus on the human α-catenin FABD (Δ886–906 or Δ866–906) bundling is no longer observed compared to the human α-catenin FABD that was not truncated (residues 660–906) (G).We observed that the HMP1/α-catenin FABD bundled F-actin E and 2A.tivation . Interestivation , B and C-catenin E. Furthe F-actin F. Howeve660–906) G.Figure D), we only observed partial decoration of F-actin as determined by 2D classification. The partial decoration consistently led to 3D reconstructions that lacked density for HMP1/α-catenin. Extensive efforts to enrich the decoration through careful curation of 2D classes did also not lead to decorated HMP1/α-catenin actin filaments.Although full-length HMP1/α-catenin binds F-actin D, we onlA).On the other hand, the isolated HMP1/α-catenin FABD bundles F-actin  which haFig. 4B). For example, Val-809 of one HMP1/α-catenin interacts with Ile-872 of a separate HMP1/α-catenin polypeptide chain.As seen for the binding of mammalian α-catenin, the carboxy terminus of HMP1/α-catenin extends to engage in hydrophobic interactions with HMP1/α-catenin of a neighboring HMP1/α-catenin B. For exC). As seen with our human α-catenin crystal structure . Specifically, Trp-711 predicted to be at the end of the first α-helix in the unbound HMP1/α-catenin FABD structure (C), that is also seen in our unbound human α-catenin structure (D). When bound to F-actin, Trp-711 fills the pocket that Trp-861 occupies in the unbound model. HMP1/α-catenin residue Trp-861 corresponds to human α-catenin residue Trp-859 (B). In the F-actin bound human α-catenin structure (E), Trp-859 fills the pocket similarly as for the equivalent HMP1/α-catenin Trp-861 (C). The extension of the second α-helix, by borrowing residues from the loop region, no longer leaves room for the loop region of the last α-helix that therefore becomes disordered in the actin-bound HMP1/α-catenin structure . This disorder is consistent with SEC-small-angle X-ray scattering (SAXS) data that showed that the FABD connects via a flexible linker (residues 630–675) to the middle domain  were not detected in 2D classification. This suggests that the middle domain of HMP1/α-catenin is the only part rigid enough to align during 2D classification, while the rest of HMP1/α-catenin is flexible A. This de domain .5h5m) (B). Similarly, the truncated murine αN-catenin structure  (C). While the truncated HMP1/α-catenin crystal structure has one polypeptide chain in the asymmetric unit (space group P 212121), the truncated αN-catenin structure has two polypeptide chains in the monoclinic (P 21) crystal. Thus, despite the different crystal contacts in the nematode versus murine middle domain structures, these can be superimposed with 1.8 Å root-mean-square deviation for 1,647 atoms. Given that the two crystal lattices resulted in similar interdomain interactions, it seems that in full-length HMP1/α-catenin, the interdomain interaction differs by ∼20 Å movements, as seen in our cryoEM structure (B and C).In comparison to our cryoEM HMP1/α-catenin structure, the truncated HMP1/α-catenin crystal structure   has two 5h5m) B. Similary 5xfl)  also hasry 5xfl) C. While tructure , B and C6upv) or murine (PDB entry 6wvt) α-catenin to F-actin (6upv) or murine (PDB entry 6wvt) α-catenin, respectively  that mediates longitudinal actin-actin interactions , 37. Ade F-actin , 18. Speectively .Molecular insights into α-catenin are crucial to understanding α-catenin regulation of cell–cell contacts. While a wealth of mammalian α-catenin structural data is available, structural insights into HMP1/α-catenin are limited. Thus, our HMP1/α-catenin studies provide significant new structural data and will additionally aid our understanding of the evolution of multicellularity in metazoans.Despite the high sequence identity amongst α-catenins from different species, only some of these proteins dimerize. Our full-length HMP1/α-catenin cryoEM structure shows that HMP1/α-catenin is a highly flexible monomeric protein. Several phosphorylation sites have been identified in HMP1/α-catenin . In contIn contrast, the phosphomimetic and nonphosphorylatable mutant of Ser-649 localized to cell–cell junctions as seen for wild type HMP1/α-catenin middle domain . This isThe lack of interdomain interactions in our full-length HMP1/α-catenin is relevant to its functions. First, the amino-terminal HMP1/α-catenin domain that binds HMP2/β-catenin at cell–cell junctions is disordered in our structure and therefore displays considerable conformational freedom. This allows HMP1/α-catenin to readily bind to partners like HMP2/β-catenin without prior activation. Similarly, we find that the carboxy-terminal FABD of HMP1/α-catenin is not interacting with the other HMP1/α-catenin domains. In contrast to mammalian α-catenin, HMP1/α-catenin is therefore constitutively active in linking HMR1/cadherin to the actin cytoskeleton. Indeed, there are also differences in the affinity of the various α-catenins with filamentous actin , 41.SEC-SAXS data showed that monomeric human αE-catenin is a compact globular structure with a maximum dimension of about 147 Å  that is Human α-catenin was shown to bind F-actin cooperatively , 44, 45.We found that full-length HMP1/α-catenin co-sedimented with filamentous actin. A S823F mutant has previously been shown to reduce the binding of HMP1/α-catenin with F-actin . This isFurthermore, the isolated HMP1/α-catenin FABD resulted in heavy F-actin bundling, as seen by our cryoEM analyses. Such bundling was not observed by full-length HMP1/α-catenin. Our findings are consistent with an earlier report that showed that HMP1/α-catenin co-sedimented with F-actin significantly less compared to the isolated HMP1/α-catenin FABD . Our cryThe carboxy terminus is disordered in our F-actin-bound FABD HMP1/α-catenin cryoEM structure and in our dimeric full-length human crystal structure. Given that carboxy-terminal truncations prevent bundling, it seems that the last ∼20 residues are responsible for bundling. The AlphaFold  model ofIt is tempting to speculate that in the full-length monomeric structure, this carboxy-terminal region might be buried in intermolecular interactions and thus prevent bundling.Although our actin-bound sample for cryoEM structure determination comprises HMP1/α-catenin residues 677 to 927, the HMP1/α-catenin amino terminus (residues 677–710) was disordered in our cryoEM map. This is consistent with the finding that HMP1/α-catenin Δ677-703 localized to cell–cell junctions as seen for wild type HMP1/α-catenin . HoweverC. elegans are just beginning to be uncovered  were cloned into the pET28 vector to have an amino-terminal octa-Histidine tag followed by a PreScission protease site immediately before the inserts using the following primers:pET28 vector:Forward: TGAGAATTCGCGGCCGCACTCGAGCACCACReverse: CATATGGGGCCCCTGGAACAGAACTTCCAGATGHuman α-catenin FABD (upper case is used for the vector and lower case for the insert):660 Forward: CTGGAAGTTCTGTTCCAGGGGCCCcatgacgatcagctgatagctggcc906 Reverse: CGGCCGCGAATTCTCAgatgctgtccatagctttgaactcgctg885 Reverse: CGGCCGCGAATTCTCAtgcccgtttaatcttggtctgtgtctcatc865 Reverse: CGGCCGCGAATTCTCActctggtgccttcatcttccatgD. discoideum α-catenin was procured as an amino-terminally tagged GST fusion with a tobacco etch virus cleavage site (Dicty stock center).The HMP1/α-catenin FABD (residues 677–927) and full-length human α-catenin (residues 1–906) were cloned into pGEX-6P-1 with an amino-terminal GST tag and a PreScission protease cleavage site by DNA Custom Cloning. The expression construct of g for 15 min and stored at −80 °C.Constructs were transformed into BL21(DE3) (Novagen) and expressed in 2 L of Luria-Bertani media at 37 °C until the absorbance at 600 nm reached 0.6 to 0.8 and induced with 1 mM isopropyl β-D-1-thiogalactopyranosides for 20 h at 25 °C. Cells were pelleted by centrifugation at 5000Escherichia coli BL21(DE3) pellets containing amino-terminally GST-tagged HMP1/α-catenin were thawed and resuspended in lysis buffer containing 400 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, and lysed by sonication (75% amplitude with 3 min processing time and 5 s on followed by 10 s off). The lysate was clarified by centrifugation  and then loaded onto a GST prep 16/10 FF chromatography column that was equilibrated with 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM EDTA, and 2 mM β-mercaptoethanol. Elution was completed with 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM EDTA, 2 mM β-mercaptoethanol, and 10 mM reduced glutathione. Peak elution fractions were pooled and dialyzed overnight in 2 L of buffer containing 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM EDTA, and 2 mM β-mercaptoethanol, after the addition of 200 μg PreScission protease. The sample was then reloaded onto a GST prep 16/10 FF column. The flowthrough containing cleaved HMP1/α-catenin was collected, concentrated to 2 mg/ml, and aliquoted into 1 ml aliquots after adding glycerol , and stored at −80 °C.To generate purified full-length HMP1/α-catenin, frozen Prior to cryoEM grid preparation, an aliquot was thawed and passed through a Superdex S200 10/300 Gl column equilibrated in 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 7.5), and 1 mM dithiothreitol. The purification of the FABD of HMP1/α-catenin was performed as described for full-length HMP1/α-catenin.D. rerio, and D. discoideum α-catenins were purified similar to HMP1/α-catenin using 1 mM dithiothreitol instead of β-mercaptoethanol. Cleavage of the GST tag for D. discoideum α-catenin was carried out with tobacco etch virus protease.Full-length human, For full-length human α-catenin, the monomer and dimer peaks were pooled separately, and a second SEC run was performed of each pool on a Superdex S200 10/300 Gl column pre-equilibrated in 20 mM tris(hydroxymethyl)aminomethane (pH 8), 150 mM NaCl, and 0.2 mM tris(2-carboxyethyl)phosphine.E. coli BL21(DE3) pellets containing amino-terminally octa-Histidine-tagged α-catenin FABDs were thawed, resuspended in 500 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), and 1 mM β-mercaptoethanol, and lysed by sonication for 3 min (5 s on and 10 s off cycles). The lysate was clarified by centrifugation at 100,000g for 45 min and filtered. The supernatant was loaded onto two HisTrap HP 5 ml columns in tandem equilibrated in a buffer containing 150 mM NaCl and 20 mM tris(hydroxymethyl)aminomethane (pH 8) on an AKTA FPLC at a flow rate of 1 ml/min. The column was then washed thoroughly with 30 column volumes of buffer containing 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), and 50 mM imidazole. Bound protein was then eluted using a gradient to 500 mM imidazole. The eluted protein fractions were pooled and subjected to overnight PreScissoin protease treatment (200 μg) with dialysis against a buffer containing 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 1 mM ethylenediaminetetraacetic acid, and 1 mM dithiothreitol. The PreScissoin protease treated α-catenin FABD proteins were concentrated and loaded onto a Superdex 75 26/60 column equilibrated in 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), and 1 mM dithiothreitol. The peak fractions were concentrated and stored at −80 °C.For purification of the human α-catenin FABD proteins , frozen We used SEC and multiangle light scattering analyses to determine the absolute masses with an Agilent 1260 Infinity HPLC with a variable wavelength detector for UV absorption monitoring which is coupled in line with Dawn-Heleos II multiangle light-scattering detector (Wyatt Technology) and OptiLab T-rex differential refractive index detector (Wyatt Technology). We loaded our samples on an analytical Superdex S200 10/300 Gl column that was pre-equilibrated with 20 mM tris(hydroxymethyl)aminomethane (pH 8), 150 mM NaCl, and 0.2 mM tris(2-carboxyethyl)phosphine, at a flow rate of 0.5 ml/min. We used bovine serum albumin (Pierce) as a standard and performed data acquisition and analyses using ASTRA software version 6.1.2, 0.5 mM β-mercaptoethanol) at 4 °C. We polymerized G-actin (at 0.6 mg/ml) by adding the polymerization buffer  to fully polymerize overnight. F-actin was stored at 4 °C for up to 2 weeks.We dialyzed rabbit skeletal muscle G-actin (50 μl at 1 mg/ml) into 500 ml of G-actin buffer (2 mM tris(hydroxymethyl)aminomethane [pH 8], 0.2 mM adenosine triphosphate, 0.2 mM CaCl2, 10 mM MgCl2, 100 mM KCl, and 10 mM adenosine triphosphate) for binding assays or in KMEI buffer , 2 mM adenosine triphosphate, and 0.5 mM ethylene glycol tetraacetic acid) and used for bundling assays. For binding assays, F-actin (15 μM) was incubated with 8 μM full-length proteins of HMP1/α-catenin, zebrafish (D. reiro) α-catenin, or slime mold (D. discoideum) α-catenin. For F-actin bundling assays, F-actin (4 μM) was incubated with 3 μM of full-length HMP1/α-catenin or 15 μM of HMP1/α-catenin FABD proteins. After incubation at room temperature for 15 min, the samples were spun at appropriate speeds  for 15 min. The supernatant and the pellet were analyzed on sodium dodecyl-sulfate polyacrylamide gel electrophoresis and the bands visualized by Coomassie R-250 staining. As controls, F-actin bundling with full-length α-catenin (residues 1–906) monomer and dimer along with α-catenin FABDs  were also analyzed.For F-actin co-sedimentation assays, rabbit muscle actin (Cytoskeleton Inc) was freshly polymerized either in Tris buffer (20 mM tris(hydroxymethyl)aminomethane [pH 8.0], 0.2 mM CaClFor the HMP1/α-catenin FABD in complex with F-actin cryoEM structure determination, C-Flat 1.2/1.3400 mesh grids were glow discharged for 60 s at 15 mA using a PELCO easiGlow (Ted Pella Inc) and mounted on Leica GP2 cryogenic plunger (65% humidity and 21 °C). First, 3.5 μl of F-actin (0.7 μM) was placed on the carbon film and incubated for 60 s and subsequently successive addition and removal of 3.5 μl of 0.6 mg/ml (21 μM) HMP1/α-catenin. The addition and removal steps were repeated twice before blotting from the backside of the grid for a total of 12 s. The grids were immediately plunge frozen in liquid ethane that is maintained at -183 K and cooled by liquid nitrogen. Lower concentrations of the HMP1/α-catenin FABD were unsuitable as they resulted in partial decoration. The grids were transferred into a Japan Electron Optics Laboratory (JEOL) cryoARM300 for screening and data collection.For the full-length HMP1/α-catenin cryoEM structure determination, an Au-Flat 1.2/1.3300 mesh grid (Protochips) was glow discharged at 15 mA for 240 s using a PELCO easiGlow (Ted Pella Inc) and taken into Leica GP2 cryogenic plunger set to 95% humidity and 4 °C. About 4 μl of 0.3 mg/ml of full-length HMP1/α-catenin was applied on the carbon film side, blotted on the carbon side for 7 s, and plunged immediately for vitrification in liquid ethane and transferred into a JEOL cryoARM300.All cryoEM data were recorded on a JEOL cryoARM300 operated at 300 kV. The illumination was set to a spot size of three and an angle of four at a magnification of 60,000. The condenser aperture was set to 50 μm with an Omega in-column energy filter, a slit width of 20 eV, and a zero-loss peak that aligned every 6 h. Electron micrographs were collected using serialEM  on a K3 Raw movies were imported into the cryoSPARC  interfacFor the FABD of HMP1/α-catenin bound to F-actin, 20,737 micrographs were collected to compensate for the high degrees of bundling observed in many micrographs and to improve the final resolution. After micrograph curation, 3576 images were selected, while the rest were removed. Since the 2D classes exhibited a high degree of decoration, a larger box size was not needed to separate unbound from bound particles. Instead, an extraction box size of 1080 pixels (399 Å) was used, and 2D classification was only used to separate suitable particles from unsuitable particles. After helical refinement, CTF refinement, and nonuniform refinement, a reconstruction of a 3D map at 3.36 Å resolution from 1,990,015 particles was achieved (EMDB entry EMD-26805), with a helical twist of −166.9°, and a helical rise of 27.72 Å.ab initio volume calculation. The final homogenous refinement yielded a 4.8 Å resolution reconstruction from 149,802 particles (EMDB entry EMD-26748).For full-length HMP1/α-catenin, a total of 2781 movies were collected, and after curation, 1294 micrographs were selected for further processing. Select 2D classes obtained from the initial round of blob picking and extraction were used to carry out the second round of template-based particle picking. The resultant 2D classes were used to select suitable particles for initial 7uuw).To determine the cryoEM structure of the HMP1/α-catenin FABD bound to F-actin, the cryoEM structure of our human α-catenin (residues 22–906) bound to F-actin was first fitted into the map using Chimera. A HMP1/α-catenin homology model was generated with SWISS-MODEL  and then5h5m) was initially docked into our cryoEM map. The structure was better fitted after chain refinement using COOT (To determine the cryoEM structure of full-length HMP1/α-catenin, the crystal structure of the middle domain of HMP1/α-catenin (PDB entry ing COOT .7uuw. The cryoEM reconstructions for the HMP1/α-catenin FABD bound to F-actin and the unbound HMP/α-catenin were deposited in EMDB with accession codes EMD-26805 and EMD-26748, respectively. All other data are within the manuscript.The coordinates for the HMP1/α-catenin FABD bound to F-actin are deposited with the Protein Data Bank, accession code This article contains The authors declare no conflict of interest with the contents of this article.