A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory mechanism of human A2ML1 and determine the structures of its native and protease-cleaved conformations. The functional inhibitory unit of A2ML1 is a monomer that depends on covalent binding of the protease (mediated by A2ML1’s thioester) to achieve inhibition. In contrast to the A2M tetramer which traps proteases in two internal chambers formed by four subunits, in protease-cleaved monomeric A2ML1 disordered regions surround the trapped protease and may prevent substrate access. In native A2ML1, the bait region is threaded through a hydrophobic channel, suggesting that disruption of this arrangement by bait region cleavage triggers the extensive conformational changes that result in protease inhibition. Structural comparisons with complement C3/C4 suggest that the A2M superfamily of proteins share this mechanism for the triggering of conformational change occurring upon proteolytic activation. A2ML1 is a human protease inhibitor belonging to the A2M protein family. In this study, the authors determine structures of A2ML1 before and after protease inhibition and investigate its mechanism of action. The physiological role of A2ML1 is unknown, although it is suspected to regulate epidermal proteases involved in keratinocyte differentiation and desquamation3. In addition, it has been implicated in the pathogenesis of paraneoplastic pemphigus4 and otitis media6. A2ML1 and other A2MF proteins share a common domain architecture. They contain eight macroglobulin (MG) domains with ~100 residues organized in two antiparallel β-sheets. The ~120 residue CUB domain (positioned between MG7 and MG8) likewise contains two β-sheets. The thioester (TE) domain is an α-helical domain of ~300 residues inserted within the CUB domain. Finally, the LNK (linker) region and bait region (BR) form a single continuous insert in the MG6 domain. In the complement members of the A2MF, the bait region is replaced by an anaphylatoxin domain separated from the LNK region in the mature complement factor by furin processing. Complement components C3, C4, and C5 contain an additional C-terminal domain that interacts with complement-specific proteases and subunits of the membrane attack complex8.Human alpha-2 macroglobulin-like 1 (A2ML1) is a monomeric member of the A2M superfamily (A2MF) of protease inhibitors and complement factors. A2ML1 is abundant in the epidermal granular layer of the skin but is also found in the placenta, thymus, and testis11. The A2MF protease inhibitors exist as functional monomers in amphibians, reptiles, birds, and rodents15, as dimers in octopus vulgaris15 and human pregnancy zone protein (PZP)16, and as tetramers such as human A2M17. With a few exceptions , A2MF proteins contain an internal thioester formed between the cysteine and a glutamine side chain in a Cys-Gly-Glu-Gln motif. After bait region cleavage or anaphylatoxin release, this thioester is exposed and can react with nucleophilic amine and hydroxyl groups on proteins and carbohydrates. As a result, the nascent activated A2MF protein may become covalently linked to the bait region-cleaving protease as seen in A2MF protease inhibitors or the complement-activating surface in the case of C3 and C4. A2ML1 can react with both amine and hydroxyl groups18, similarly to C3 and C4B19, as the nascent thioester is attacked by a nearby histidine residue to form an acyl-imidazole intermediate. The acyl-imidazole group has an increased reactivity that allows it to react with hydroxyl groups in addition to amine groups.A2MF protease inhibitors utilize a unique trapping mechanism that pseudo-inhibits proteases regardless of their catalytic class. Trapped proteases remain catalytically active but are prevented from cleaving protein substrates through steric hindrance imposed by the inhibitor following a proteolytically triggered conformational change21 provide a detailed understanding of their conformational change following proteolytic cleavage. In contrast, A2MF protease inhibitors are relatively poorly structurally characterized, excepting a low-resolution structure of methylamine-treated A2M that suggests a subunit conformation different from that of complement C3b and C4b22 and a recently determined cryo-EM structure of a native tetrameric ovostatin (which strongly resembles A2M) from the frog X. laevis23. Structures for two bacterial A2MF protease inhibitors, thought to originate from horizontal gene transfer from animals, have been determined but show that these proteins are structurally quite distinct from their animal counterparts26.Structural studies of complement factors such as C3 and C4To provide a structural explanation for the unique mechanism of protease inhibition by A2MF proteins, we determined structures of native A2ML1 and three different protease-cleaved forms of A2ML1, and assessed the ability of an A2ML1-bound protease to cleave substrates. Our results demonstrate that the functional inhibitory unit of A2ML1 is a monomer that requires the formation of a covalent bond between a protease and A2ML1 in order to achieve protease inhibition by steric hindrance. Based on our structures, we propose that disordered regions in A2ML1 provide a pivotal contribution to protease sequestration and we suggest a unified mechanism for the conformational change occurring upon proteolytic activation of A2MF proteins.14, whereas this is not required by tetramers like A2M. A2ML1 is present as a monomer in human skin and remains monomeric after bait region cleavage by proteases , gelatin (average molecular weight of 100 kDa), and A2M (720 kDa)  Fig. . Overall18. To assess A2ML1 cleavage by other proteases, we incubated A2ML1 with titration series of chymotrypsin, porcine pancreatic elastase (PPE), and trypsin in the presence and absence of BAPN  located between the ANA domain and the C-terminal half of the MG6 domain passes through a channel formed by the LNK region, the MG2 and MG3 domains, and the MG2-MG3 linker. Upon cleavage, the Nt-α′ region retracts from this channel and relocates to a position between the MG6 and MG7 domains. In A2ML1, the equivalent of Nt-α′ corresponds to the residues C-terminal of the scissile bond in the bait region and for this reason we refer to it as the BR-C region. In the structures of TEP1r/s, the corresponding region is not modeled, suggesting a higher mobility or that it is disordered. In both the EM and the X-ray structures of native A2ML1, it is not possible to model residues His696-Asp726, suggesting substantial flexibility of the intact bait region in accordance with its accessibility toward proteases  either could not be modeled or have a weak electron density. These disordered regions, as well as glycans on Asn328 and Asn857, are oriented toward the protease-binding cavity 26 have been determined. Both bacterial proteins have two additional N-terminal MG domains and significantly altered MG-ring configurations compared to the animal A2MFs. Comparison of the native Salmonella A2MF and cleaved ECAM structures reveals a very large reorganization of the MG ring after bait region cleavage, whereas the MG ring is mostly static in animal A2MFs. The TE domain in cleaved ECAM is located in an extended position where it does not directly contact the MG ring and may be stabilized by the ECAM equivalent of the MG8 domain, which has a defined position in the structure associating with the MG ring. Furthermore, in the bacterial proteins, the additional N-terminal MG domains that are not present in animal A2MFs may contribute to shielding conjugated proteases. In contrast to A2ML1 which sequestered proteases from medium-sized substrates (≥24 kDa) in this study, ECAM only prevented trapped proteases from cleaving very large substrates (≥160 kDa)25. The rat A2MF protease inhibitor A1I3 can also sequester proteases from medium-sized proteins (≥22 kDa)14 and although its structure has not been determined, its sequence indicates it largely resembles A2ML1. These major structural and functional differences indicate that monomeric protease inhibitors from bacteria and animals are mechanistically distinct despite the dependence of both on covalent protease conjugation.Based on these observations and our biochemical characterization of A2ML1, we propose a model of protease inhibition which requires both thiol ester-mediated conjugation for protease trapping and a dynamic mesh of exposed flexible regions that help to shield the trapped protease from substrates. This model provides a possible explanation for how an A2MF monomer can inhibit proteases without completely surrounding a trapped protease. Could this mechanism extend to other monomeric relatives of A2ML1? With respect to insect TEP proteins, no protease-inhibiting ability has been demonstrated, and these proteins are believed to have complement-like functionsIn native A2ML1, residues Arg730-Pro734 at the C-terminal end of the bait region (BR-C) form a plug within a channel formed by the MG2 and MG3 domains on either side, the MG2-MG3 linker from above, and the first α-helix in the LNK region from below Fig. . Based o41, along with other mutations which we predict to destabilize domain interfaces  and carried out according to the Declaration of Helsinki Principles.Streptococcus aureus, thermolysin from Geobacillus stearothermophilus, and porcine pancreatic elastase (PPE) were from Sigma-Aldrich, while human neutrophil elastase (HNE) and human cathepsin G (CatG) were from Athens Research & Technology. Plasminogen was purified from human plasma as previously described43 and activated by incubating with urokinase at a 1:100 w/w ratio at 37 °C for 2 h. A1I3 was purified as previously described from rat plasma14 and A2M from human plasma44. The human skin sample was obtained from a healthy donor after skin removal surgery, and the Declaration of Helsinki protocols were followed.Bovine pancreatic trypsin, bovine pancreatic α-chymotrypsin, endoproteinase C (GluC) from 18. A2ML1t and A2ML1t-fMG8 plasmids were prepared by mutagenesis performed by Genscript. All recombinant A2Ml1 sequences are given in Supplementary Note 6 cells/ml. After incubation for 96 h, the culture supernatant was collected and dialyzed overnight at 4 °C into 0.1 M sodium acetate, 0.8 M NaCl, pH 7.0. The dialyzed medium was filtered and applied to a HiTrap Chelating HP column with immobilized Zn2+. Bound A2ML1 was eluted with 0.1 M sodium acetate, 0.15 M NaCl, 50 mM EDTA, pH 8, and the eluate was dialyzed into 20 mM HEPES, pH 7.4 overnight at 4 °C. The dialyzed sample was subjected to anion exchange on a HiTrap Q column and A2ML1 was eluted with a gradient from 0 to 40 mM NaCl. Fractions containing A2ML1 were pooled, concentrated, and subjected to SEC on a Superdex 200 increase 10/300 GL column equilibrated in HBS.For expression of wildtype A2ML1, a gene encoding its canonical amino acid sequence (Uniprot ID A8K2U0) was synthesized and cloned into the pcDNA3.1(+) plasmid, as described previouslyA total of 50 µg of A2ML1 and A1I3 and 20 µg of native A2ML1t and A2ML1-CC were subjected to calibrated SEC on a Superdex 200 Increase 10/300 GL column  equilibrated in HBS.45. Denaturing SDS−PAGE was performed using homemade 5–15% (w/v) acrylamide gels and the discontinuous ammediol/glycine buffer system46. Samples were boiled for 5 min in SDS (1%) and 35 mM dithiothreitol (DTT). Gels were stained with Coomassie Brilliant Blue.Native pore-limited PAGE was run using homemade TBE buffer  gels with a 10–20% acrylamide gradient. Gels were run overnight at 100 V in recirculating TBE buffer, as previously describedA 0.3 mg sample of human skin was vortexed with 0.1% NP40, incubated for 30 min rotating, and sonicated in a water bath for 15 min. The extracted proteins were run on a native pore-limited PAGE gel, and the gel lane was cut into 10 equally sized slices prior to in-gel digestion and LC-MS/MS analysis. The gel slices were shrunk using acetonitrile, reduced (0.1 M DTT), and alkylated (0.2 M iodoacetamide) in 6 M Urea, 0.1 M ammonium bicarbonate, pH 8. The sample was diluted x6 using 0.1 M ammonium bicarbonate and digested with 25 ng/ml MS grade trypsin overnight at 37 °C (Thermo Fisher Scientific). The digested peptides were purified using homemade C18 stage tips and analyzed by LC-MS/MS. Online reverse-phase high-performance liquid chromatography (RP-HPLC) was done on an EASY-nLC 1200 (Thermo Fisher Scientific) with a 5–45% gradient of acetonitrile (ACN) over 30 min. Mass spectrometry was performed on a Q Exactive Orbitrap (Thermo Fisher Scientific) running in data-dependent acquisition mode with 70,000 resolution at the MS1 level and 35,000 resolution at the MS2 level; peptides were fragmented by high-energy collision dissociation (HCD) at 27% normalized collision energy. The data were searched using the Mascot search engine (version 2.5) with fully tryptic protease cleavage, using methionine oxidation as variable modification, with mass tolerances of 10 ppm for precursors and 0.2 Da for MS2 fragment ions, a minimum peptide mass of 400 Da, and a false-discovery rate of 0.05 at both peptide and protein levels. The taxonomy was set to Homo Sapiens and the SwissProt database was used.Recombinant A2ML1 was digested with trypsin alone or both trypsin and chymotrypsin in solution. Digested samples were purified using homemade C18 stage tips before analysis by LC-MS/MS. Online RP-HPLC fractionation using an EASY-nLC 1200 (Thermo Fisher Scientific) was performed with a 5–45% gradient of acetonitrile (ACN) over 30 min. Mass spectrometry was performed on an Orbitrap Eclipse Tribrid (Thermo Fisher Scientific) running a glycan-triggered MS2(HCD)_MS2(EThcd) data-dependent acquisition method with a resolution of 120,000 on the MS1 level and 60,000 on both MS2 levels. HCD fragmentation used stepped collison energies  whereas EThcD used automatically adjusted ETD fragmentation augmented with 10% HCD. Data were searched with the Byonic search engine (Version 3.7.13. Protein Metrics) with a standard human glycosylation database and a PEP 2D score threshold/cut-off of 0.05.Digestions with chymotrypsin, PPE, and trypsin were done with up to a 1:1 molar ratio of protease to A2ML1. All of the reactions were carried out in HBS with and without 50 mM BAPN. The reaction mixes were incubated for 15 min at 37 °C, after which digestion was stopped by inhibiting with 2 mM phenylmethanesulfonyl fluoride (PMSF) for 15 min before SDS-PAGE analysis.Digests with thermolysin, chymotrypsin, and HNE were carried out with a molar ratio of 1:10 of protease to A2ML1 in the presence of a 0–0.2 M titration series of BAPN. The reactions were incubated for 15 min at 37 °C before residual protease activity was inhibited by adding 2 mM PMSF or 10 mM EDTA in the case of thermolysin before SDS-PAGE analysis.To identify bait region cleavage sites by different proteases using Edman degradation, samples of A2ML1 incubated with a 1:1 molar ratio of trypsin, thermolysin, HNE, cathepsin G, V8, PPE, or chymotrypsin were separated by reducing SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The C-terminal bait region cleavage bands were cut out and applied to TFA-treated glass fiber membranes. Automated Edman degradation was performed using a PPSQ-31B protein sequencer (Shimadzu Biotech) with in-line phenylthiohydantion analysis on an LC-20AT HPLC system. Data were obtained using Shimadzu PPSQ-31B software and the sequences were determined manually from the UV 269 nm chromatograms.2 fragment ions were selected; the false-discovery rate was 0.05 at the peptide and protein level. The following variable modifications were considered; methionine oxidation, proline oxidation, and glutamine/asparagine deamidation along with the following variable modifications of the N-terminal; acetylation, met-loss, and acetylation combined with met-loss. Cleavage sites that were identified more than once were reported.To identify bait region cleavage sites by different proteases using LC-MS/MS, A2ML1 was incubated with a 1:2 molar ratio of trypsin, thermolysin, HNE, cathepsin G, V8, PPE, or chymotrypsin to A2ML1. The liberated peptides were purified using homemade C18 stage tips and analyzed by LC-MS/MS on a Q Exactive Orbitrap (Thermo Fisher Scientific), using the same methods as described for the identification of A2Ml1 in skin extract. The data were searched using Proteome Discoverer v. 2.5 with no enzyme in the case of PPE, thermolysin, and cathepsin G, while the relevant protease specificity was selected for GluC, chymotrypsin, and HNE. Up to two missed cleavages were allowed, and mass tolerances of 10 ppm for precursors and 0.02 Da for MS2 for 15 min at 37 °C. All samples were subsequently incubated with 40 µl of 0.8% azocasein (Sigma-Aldrich) for 60 min (thermolysin), 120 min (chymotrypsin), or 240 min (HNE) at 37 °C before trichloroacetic acid (TCA) was added to a final concentration of 2% to terminate the reactions and precipitate intact substrate. The precipitated substrate was removed by filtration using a MultiScreen Solvinert Filter plate (Millipore) by centrifugation at 500 × g for 3 min. The filtrate was collected in a 96-well Nunclon Delta Surface plate (ThermoFisher Scientific) and neutralized by addition of 2 M Tris-HCl, pH 8.8. The residual proteolytic activity was determined by measuring the absorbance at 440 nm using a FLUOstar Omega plate reader. All reactions were performed in triplicates. The inhibition of thermolysin, chymotrypsin, and HNE were investigated using a fluorescently labeled gelatin substrate, DQ™ Gelatin From Pig Skin (Invitrogen). 8.0 pmol of chymotrypsin, 1.4 pmol of thermolysin, or 1.7 pmol of HNE were reacted with a 0–8 times molar excess of A2ML1 in 50 mM HEPES, 100 mM NaCl, 5 mM CaCl2 pH 8 for 15 min at 37 °C. The gelatin substrate was added to a final concentration of 0.1 mg/ml and the fluorescence (excitation at 485 nm and emission at 520 nm) of the digestion products of DQ gelatin were measured in a FLUOstar Omega plate reader (BMG LABTECH) after 4 min (thermolysin), 10 min (chymotrypsin), or 20 min (HNE) at 37 °C. All reactions were performed in triplicates.A total of 5.8 pmol of thermolysin was reacted with 0–23.1 pmol of A2ML1 with and without 50 mM BAPN, 12 pmol of chymotrypsin was reacted with 0–67.9 pmol of A2ML1, and 17 pmol of HNE was reacted with 0–136 pmol of A2ML1 in 120 µl of 200 mM HEPES pH 7.8, 20 mM CaCl47 built 20180808. Phenix programs from the 1.19.2-4158 version used for crystallographic and cryo-EM structural work. The structure was determined in phenix.phaser48 using two complementary density search models covering MG1-6 and CTM8, respectively, excised from a preliminary 3.8 Å cryo-EM map. After density modification with phenix.density_modification49, a near complete EM structure of native A2ML1 was placed domain-wise in the two molecules within the asymmetric unit present in the crystallographic map. The resulting model was in an iterative manner manually rebuild in Coot 0.9.350 and refined in phenix.refine51 using positional, TLS, non-crystallographic symmetry restraints, and grouped B-factor refinement.Native A2ML1 was incubated with a 1:10 (w/w) ratio of bacterially expressed GST-tagged PNGase F, MBP-tagged Endo F2, and MBP-tagged Endo F3 to A2ML1 at room temperature for 24 h. The sample was concentrated prior to SEC on a Superdex 200 10/300 column. Fractions from SEC containing pure A2ML1 were pooled and concentrated to 5–6.5 mg/ml followed by centrifugation for 5 min immediately prior to crystallization. A2ML1 was screened for crystallization in sitting-drop vapor diffusion experiments using the commercial screens PEGRx and PEGIon (Hampton research). Drops of 150 nl A2ML1 mixed with 150 nl reservoir solution were generated with an Oryx4 crystallization robot (Douglas Instruments) and equilibrated against 50 µl reservoir solution in 96-well Swissci MRC crystallization plates at room temperature. The final optimal reservoir condition contained 0.1 M potassium phosphate dibasic, 10% PEG 3350 pH 9.2 in drops of 0.9 µl A2ML1 mixed with 0.1 µl seeds and 1 µl reservoir buffer. Crystals were soaked into the reservoir solution added 25% (v/v) ethylene glycol as cryoprotectant followed by flash cooling in liquid nitrogen. Data collection with MXCUBE3 from flash-frozen crystals was performed at BioMAX  at 100 K and processed with XDSRecombinant native A2ML1 was concentrated to 1 mg/ml using a Vivaspin 2 centrifugal concentrator unit with a 30-kDa cut-off  before grid preparation for cryo-EM. To generate the A2ML1-CC and A2ML1-CA samples 600 µg of A2ML1t was incubated overnight at room temperature with a 1:1 molar ratio of A2ML1 to TEV-P in HBS with or without 200 mM BAPN. The cleaved A2ML1 samples were prepared for hydrophobic interaction chromatography on a HiTrap Phenyl HP 1 ml column by addition of 1.6 M sodium sulfate pH 7.4 to a final concentration of 800 mM. The samples were loaded onto the column and subsequently eluted with a gradient from 560 mM sodium sulfate pH 7.2 to 0 mM sodium sulfate pH 7.2.3, 0.5 M NaCl pH 8.3) followed by incubation with 1.3 mg/ml of TEV-P in coupling buffer for 1.5 h. Beads were subsequently washed in coupling buffer and incubated with blocking buffer (0.1 M Tris-HCl pH 8) for 2 h followed by washing 6 times in coupling buffer. For cleavage, 500 µl TEV-P coupled beads were washed in HBS 3 times, incubated with 5 mM DTT in HBS for 15 min and washed in HBS 3 times. An amount of 800 µg of A2ML1t was incubated with the beads in HBS in the presence of 100 mM BAPN for 2 h. The cleaved A2ML1 sample was subjected to SEC on a Superdex 200 increase 10/300 GL column equilibrated in HBS. Fractions containing cleaved A2ML1 were pooled and concentrated prior to cryo-EM.To prepare a resin with immobilized TEV-P for preparation of the A2ML1-CE sample, 0.5 g of CNBr-activated Sepharose 4B beads (Sigma-Aldrich) were incubated with 1 mM HCl for 30 min at room temperature. The beads were washed 5 times in 1 mM HCl followed by 1 time in coupling buffer (0.1 M NaHCOCopper grids CF-1.2/1.3-3Cu-50 (Protochips) were glow discharged with a current of 15 mA for 45 s using a GloQube (Quorum Technologies) system. Cryo-EM samples were prepared with a Mark IV Vitrobot system (Thermo Fisher Scientific). Briefly, 3 µL of sample were applied to each grid, the temperature during grid preparation was 4 °C and the relative humidity in the chamber was set to 100%. The blot force was 0 or 1 and the blot time 4.5 s. Data collection using the EPU 2.7 software was done on a Titan Krios G3i (Thermo Fisher Scientific) equipped with a K3 detector (Gatan) with a Bioquantum (Gatan) energy filter (slit width of 20 eV) and a C2 aperture with a 50 μM diameter, spot size 3 and a beam diameter of 0.98–1.12 μm. Data were collected in counted super-resolution mode with gain correction on the fly followed by 2x binning in EPU resulting in movies output at physical pixel size (0.647 Å/pixel at the magnification used). For processing of all the datasets, the collected movies were imported into CryoSparc v3 (Structura Biotechnology Inc), with the following parameters: 300 kV accelerating voltage, 2.7 mm spherical aberration, 0.1 amplitude contrast fraction, 130,000 nominal magnification . All subsequent processing steps were carried out in CryoSparc v3. Key parameters and statistics for data collection are presented in Supplementary Table In general, the workflow for image analysis and 3D reconstruction was the same for all four data sets. The specific steps are described in Supplementary Fig. 52 and rebuilt with Coot in an iterative manner until convergence. Starting models for the A2ML1-CE monomer and the A2ML1-CA dimer were generated from the structure of A2ML1-CC and first fitted by rigid-body refinement with phenix.real_space_refine into the relevant map after which the model was fitted in Coot and refined again in an iterative manner until convergence. A starting model for native A2ML1 was constructed from domains taken from the A2ML1-CC structure combined with a homology model for the TE domain based on the structure of native C3 (pdb entry 6RU553) and the crystal structure of the isolated MG8 domain of bovine A2M (pdb entry 1AYO54). The model was refined and rebuilt as described for A2ML1-CC. Model quality was evaluated with phenix.validation_cryoem55. The structures of native A2ML1 and A2ML1-CE were refined to a slightly lower maximum resolution compared to the map resolution estimated by cryosparc as presented in Supplementary Table An atomic model for A2ML1-CC was generated by homology modeling starting from the structures methylamine-treated A2M in pdb entry 4ACQ (22). After overall fitting in Chimera-X, each domain of the starting model was placed by rigid-body refinement in Coot and manually rebuilt to the density. The model was refined in phenix.real_space_refine using rigid-body refinement, positional refinement and grouped B-factor refinementhttps://pymol.org/pymol.html, or Chimera-X 1.1 or 1.356. An intact model of native A2ML1 was prepared starting from the cryo-EM structure of A2ML1. Unmodelled regions were adopted from the X-ray and alphafold257 structures of A2ML1, and the bait region was manually adjusted to the putative path. Proper geometry was enforced by real-space refinement of distorted regions in Coot. To create the equivalent structure of protease-cleaved A2ML1, missing regions in A2M-CC were replaced as done for the native structure, where after residues 691–710 were rotated to a more exposed position to illustrate bait region collapse. Finally, the conformation of LNK residues not observed in A2ML1-CC was modified by hand in Coot compared to their conformation in the model of full native A2ML1.All Figures were prepared with PYMOL 2.3.0, 58 searches against annotations from genome sequences of the placazoa Trichoplax (Trichoplax adhaerens), the cniderias swiftia (Swiftia exserta) and starfish (Asterias rubens), the deuterostomes littorina snail (Littorina littorea) and horseshoe crab (Limulus polyphemus), the urochordate vase tunicate  and the nematode C. elegans (Caenorhabditis elegans). Only full-length coding sequences were considered. Using CLC Main Workbench 21.0.5 (QIAGEN), the sequences were aligned with Clustal Omega59. A maximum-likelihood phylogeny was reconstructed on the multiple sequence alignment. Bootstrap values from 1000 bootstrap replicates were computed. To investigate A2MFs in Porifera, tblastn searches limited to nucleotide entries assigned to Porifera taxonomy (taxid: 6040) were conducted using the Trichoplax CD109-like protein (NCBI: XP_002111588.1) as query. Searching non-redundant (NR) and whole genome shotgun contigs (WGS) databases gave no results, whereas transcriptome shotgun assembles . Using these hits as queries in blastx searches of the entire NR database revealed that these sequences most likely originate from other taxa  and therefore do not support the presence of A2MFs in Porifera. No Porifera reference genome has been sequenced to date. To investigate A2MFs in fungi and plant kingdoms, tblastn searches limited to nucleotide entries assigned to Fungi (taxid:4751) and green plants (taxid:33090) were conducted, again using the Trichoplax CD109-like protein (NCBI: XP_002111588.1) as query. No hits were returned for fungi. The five hits returned for plants  were then used as queries in blastp searches of the entire NR database. This analysis revealed that two sequences from land plants  most likely originate from metazoan taxa (mites) and are hence not supporting the presence of A2MPs in land plants. However, the three hits originating from green algae  most likely represent authentic sequences. The lack of wider taxonomic presence of A2MFs in Mamiellales genomes indicates that these sequences probably have been obtained through horizontal transfer with metazoan origin.Human A2MF protein sequences were downloaded from NCBI. Human A2ML1 (XP_011518868) and human CD109 (Q6YHK3) were used as queries for NCBI blastpFurther information on research design is available in the Supplementary InformationReporting Summary


Nuclear receptor subfamily 1 group H member 4 (NR1H4) have been reported in various cancer types, however, little is known about the clinical values and biological function in clear cell Renal cell carcinoma (ccRCC).The expression pattens of NR1H4 in ccRCC were investigated in clinical specimens, cell lines and publicly‑available databases. Cell Counting Kit-8 (CCK-8), colony formation, 5-ethynyl-2' -deoxyuridine (EdU), transwell and cell wound healing assays were performed to assess the biological functions of NR1H4 in 786-O ccRCC cells. Gene set enrichment analysis (GSEA), Flow Cytometry, quantitative real‐time PCR (qRT-PCR), western blot and immunofluorescence were performed to explore the molecular mechanism of NR1H4 in ccRCC. We explored the early diagnostic value, prognostic value, genetic mutation and DNA methylation of NR1H4 by a comprehensive bioinformatics analysis based on the data published in the following databases: The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan‐Meier Plotter, Gene Expression Profiling Interactive Analysis (GEPIA), UNIVERSITY OF CALIFORNIA SANTA CRUZ Xena (UCSC Xena), cBio Cancer Genomics Portal, MethSurv, SurvivalMeth and The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Its correlation with tumor-infiltrating immune cells in ccRCC was analyzed by Tumor Immune Estimation Resource 2.0 (TIMER2.0) and Tumor Immune System Interactions Database (TISIDB).In this study, NR1H4 was found to be highly expressed in ccRCC tissues and ccRCC cell lines. Knockdown of NR1H4 significantly suppressed cancer cell proliferation, migration and invasion. Mechanistically, tumor‐associated signaling pathways were enriched in the NR1H4 overexpression group and si-NR1H4 could induce the downregulation of Cyclin E2 (CCNE2). By bioinformatics analysis, NR1H4 was identified as highly expressed in stage I ccRCC with a high diagnostic accuracy (area under the receiver operating characteristic curve > 0.8). Genetic alteration and DNA methylation of NR1H4 were significantly associated with prognosis in ccRCC patients. Moreover, NR1H4 expression associated with immune cell infiltration levels in ccRCC, which provides a new idea for immunotherapy.Our study indicated that NR1H4 might be a potential tumor biomarker and therapeutic target for ccRCC which could promote cancer cell proliferation, migration and invasion via regulating CCNE2.The online version contains supplementary material available at 10.1186/s12885-022-10087-4. Global NR1H4, also called farnesoid X receptor (FXR) is a transcription factor belonging to the superfamily of nuclear receptors , 6. NR1HAbnormal expression of cyclins affects the cell cycle and eventually leads to the progression of cancer. CCNE2 and its associated catalytic partners, cyclin-dependent kinase 2 (CDK2), have important functions in cellular biological processes , 18. CCNIn the present study, we reported that NR1H4 was overexpressed in ccRCC and high expression of NR1H4 is important to promote tumor progression. Moreover, we first assessed its correlation with clinical prognosis, early diagnosis and infiltrating immune cell in ccRCC through bioinformatics analysis. Our data revealed a novel regulator in ccRCC development and implicated the clinical significance of NR1H4.https://portal.gdc.cancer.gov), including 538 tumor samples and 72 adjacent normal tissues' transcriptome data and clinical information of the corresponding patients.RNA-seq data and clinical information for ccRCC were obtained from TCGA (https://www.ncbi.nlm.nih.gov/geo) and used for the validation. Table Four sets of microarrays  were downloaded from the GEO database (http://timer.cistrome.org/), a web server for comprehensive analysis of tumor-infiltrating immune cells [TIMER2.0 , an analysis tool containing RNA sequence expression data of 9,736 tumors and 8,587 normal tissue samples [GEPIA , an interactive web resource [UALCAN  and UCSC Xena (https://xena.ucsc.edu/) were used to assess the prognostic value of NR1H4 in ccRCC [The Kaplan‐Meier Plotter , a comprehensive web resource for interactive exploration of multidimensional cancer genomic datasets [The cBio Cancer Genomics Portal  was used to assess the DNA methylation level of NR1H4 and SurvivalMeth  was used to assess the prognostic value of the DNA methylation of NR1H4 in patients with ccRCC [MethSurv . HK-2 was cultivated in DMEM medium  supplemented with 10% fetal bovine serum (FBS)  and ccRCC cells were cultured in RPMI 1640 medium  supplemented with 10% FBS. All cells were cultured in the same humidified atmosphere (37 °C with 5% CO2). 786-O cell line was transfected by NR1H4-specific siRNA for 6 h. Meanwhile, nontargeting siRNA  was used to transfect 786-O cells as a negative control. The siRNA transfections were performed using Lipo6000 transfection reagent , according to the manufacturer’s protocol. After 48 h, cell experiments were performed.Total RNA was isolated using the TRIzol reagent  and was reversely transcribed to cDNA with the PrimeScript™ RT Reagent Kit with gDNA Eraser . qRT-PCR was performed on the Roche LightCycler 480 detection system with TB Green Premix Ex Taq II  and gene expressions were normalized to GAPDH. The quantitative analysis was calculated by using 2 − ΔΔCt method. The primers were all listed in Additional file 1: Table SFor western blot, proteins were separated on 10% SDS-PAGE gels (50 mg/lane) and then transferred to PVDF membranes . The membranes were blocked with 5% non-fat milk in TBST buffer  for 1 h and incubated with primary antibodies overnight at 4 °C. Primary antibodies used here were monoclonal mouse antibody against NR1H4/FXR , monoclonal mouse antibody against Cyclin E2 , polyclonal rabbit antibody against CDK2  and polyclonal rabbit antibody against GADPH . After extensive washing with TBST buffer, the membranes were incubated with anti-mouse  or anti-rabbit  IgG secondary antibody at room temperature for an additional 1 h. Protein bands were scanned by the ChemiDoc™ XRS + system and analyzed using an Image Lab software .6 cells/ml) with different pretreatments were seeded into 6‐well plate and fixed with 75% ethanol overnight. Cells were then resuspended with cold PBS and stained with 100 μg/ml of RNase A and 50 μg/ml of PI for 30 min in the dark. CytoFLEX  was used to analyze the DNA content of cells.Cell cycle analysis was performed by flow cytometry with PI staining. 786-O cells (1 × 10For immunohistochemistry (IHC), the paraffin-embedded tissues were cut into 5 μm sections and were incubated with specific antibodies for NR1H4  overnight at 4 °C, then incubated with secondary antibody for 30 min at 37 °C, followed by the addition of a stain. For immunofluorescence, the cells of different treatments were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100/PBS. After washing with PBS, they were blocked with 10% goat serum for 1 h at room temperature and incubated with mouse monoclonal antibody against Cyclin E2  for 2 h. After several washes with PBS, the cells were treated with the secondary antibody (goat anti-rabbit) for 1 h at 37 °C. The nuclei were stained with DAPI. Images were collected under microscope.For CCK8 assay, cells were seeded in a 96‐well plate at the density of 2,000 cells per well. The cell viability was detected at four selected time points . CCK‐8 solution (10μL) was added to each well at indicated times and incubated for another 3 h. The optical density values were detected at 450 nm .For colony formation assay, cells were seeded into 10 cm plate with 1000 cells/well. When there were at least 50 cells under microscope for single clone after around 2 weeks, clones were fixed using 4% paraformaldehyde and stained using 0.1% crystal violet. After washed and aired, clones were pictured.4 cells/well) were seeded in 24-well plates. Subsequently, cells were incubated with EdU for 2 h, fixed with 4% paraformaldehyde and stained the nuclear with Hoechst. Images were collected under microscope.For EdU assay, the EdU kit  was used. 786-O cells .For transwell assay, 1 × 10For wound healing assay, cells were seeded into 6‐well plates. After the cells reached 80%‐90% confluence, the cell layer was scratched with a 10 µl pipette tip and the medium containing 10% FBS was replaced with a serum-free medium. The wounds were observed and imaged at 0 and 24 h after wounding.GSE167093 with a functional gene set file (c2.cp.kegg.v7.4.symbols.gmt) was analyzed by GSEA to obtain pathways enriched by NR1H4. High (top 50%) and low (bottom 50%) NR1H4 expression groups were divided according to the median expression of NR1H4. Gene sets with nominal p-value less than 0.05 and FDR less than 0.25 were considered of statistically significant.All data were processed using the R software (version3.6.0), GraphPad 8.0, and SPSS version 23.0 (SPSS). Paired t‑test, unpaired t-test and one‐way ANOVA followed by Tukey post hoc test were used to compare the expression of NR1H4 in different groups. A Chi-square test was performed to analyze the correlation of NR1H4 expression and clinicopathological factors. The diagnostic value of NR1H4 in lung cancer was revealed by receiver operating characteristic (ROC) curves. Univariate and multivariate survival analyses were executed using the Cox proportional hazards regression model. All experiments were performed at least three times; data are presented as mean ± SD. P values less than 0.05 were considered statistically significant.Abnormal expression of NR1H4 has been reported in various cancers, 31. HowAccording to previous reports, inhibition of NR1H4 has an anti-cancer effect . To explp = 0.0041), CCND1 , CCND2  and CCNE2  expression. The results of qRT-PCR indicated that si-NR1H4 could induce the downregulation of CCNE2 rather than other cyclins , GSE46699 with AUC = 0.7953  and GSE40435 with AUC = 0.8623  . These results revealed that NR1H4 possesses a high diagnostic potential in differentiating ccRCC patients from normal individuals, even for the early stages of ccRCC. To investigate the prognostic value of NR1H4, the Kaplan–Meier Plotter database, GEPIA database and UCSC Xena database were used. However, we found patients with higher expression of NR1H4 did not have poorer OS  or DFS   and PFS (P = 7.887e-4) of ccRCC patients  and neutrophil  infiltration levels   , neutrophil  and active dendritic cell  infiltration levels    The correlations between NR1H4 expression and immune infiltration levels of ccRCC by TISIDB database analysis. (B) The correlations between NR1H4 expression and macrophage infiltration levels by XCELL algorithm analysis.Additional file 4. Original blots in the manuscript.


Catha edulis Forsk) plant. Khat was found to inhibit major phase I drug metabolizing cytochrome P450 (CYP) enzyme activities in vitro and in vivo. With the upsurge of khat consumption and the potential use of cathine to combat obesity, efforts should be channelled into understanding potential cathine-drug interactions, which have been rather limited. The present study aimed to assess CYPs activity and inhibition by cathine in a high-throughput in vitro fluorescence-based enzyme assay and molecular docking analysis to identify how cathine interacts within various CYPs’ active sites. The half maximal inhibitory concentration (IC50) values of cathine determined for CYP2A6 and CYP3A4 were 80 and 90 μM, while CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2 and CYP3A5 showed no significant inhibition. Furthermore, in Ki analysis, the Lineweaver-Burk plots depicted non-competitive mixed inhibition of cathine on both CYP2A6 and CYP3A4 with Ki value of 63 and 100 μM, respectively. Cathine showed negligible time-dependent inhibition on CYPs. Further, molecular docking studies showed that cathine was bound to CYP2A6 via hydrophobic, hydrogen and π-stacking interactions and formed hydrophobic and hydrogen bonds with active site residues in CYP3A4. Both molecular docking prediction and in vitro outcome are in agreement, granting more detailed insights for predicting CYPs metabolism besides the possible cathine-drug interactions. Cathine-drug interactions may occur with concomitant consumption of khat or cathine-containing products with medications metabolized by CYP2A6 and CYP3A4.Cathine is the stable form of cathinone, the major active compound found in khat ( Catha edulis Forsk plant, which is also commonly known as khat. Khat is a perennial shrub cultivated in khat-belt countries such as in Africa and the Middle East, that are ingested not to gain nutritive values but to attain psychostimulatory effects. Khat was traditionally used in social gatherings in Yemen and widely cultivated as a source of income replacing coffee cultivation despite debates that khat cultivation drains foreign investment. Khat chewing is a tradition in khat-belt countries with each khat sessions lasting 3 to 4 hours with 100-200 g of leaves chewed, which induces mild euphoria and excitation in its users. In the early 1930s, Wolfes identified cathine phenylpropanolamine) as norpseudoephedrine, which was one of the khat alkaloids that contribute to the pharmacological effects of khat leaves. It was reported that in every 100 g of fresh khat, there were 36 mg of cathinone, 120 mg of cathine and 8 mg of norephedrine present. Young stems and flowers of khat plants contain 1-phenyl-1,2-propanedione, cathinone, cathine and norephedrine. Cathinone reductase present in khat was accountable for reducing cathinone to cathine in the presence of NADPH. However, the quantity of cathine depends on the type of khat in which green khat contains a higher amount of cathine and norephedrine than red khat. Besides khat plant, cathine is also found in anorectic products. Cathinone and cathine shares closely similar chemical structures to amphetamine. Cathine was claimed to be 7-10 times less potent than cathinone but possesses a longer duration of action.Cathine (d-norpseudoephedrine (NPE)) is one of the major constituents found in the  As compared to cathinone, cathine has a slower onset of action and may not be as lipophilic to enter the central nervous system, which to some extent explains the slower metabolism of cathine. Oral administration of cathine (6.5 mg/kg) may affect adrenocortical function and was found to produce rapid synchronized cell death in human leukaemia cell lines and peripheral blood leucocytes. With its milder psychostimulatory effects, a higher dose of cathine is needed to exert its effects, and this causes severe adverse systemic effects and the inhibition of noradrenaline uptake.12 In recent years, khat use was reported in England, Wales, Rome, Amsterdam, Canada, Israel, Australia, New Zealand and the United States. Khat is chewed by individuals idling on streets in Europe accompanied sometimes by alcoholic beverages and other drugs.Over the past decades, cathine has received little experimental attention. Cathine acts as a central stimulant (indirect sympathomimetic) and an inhibitor or monoamine oxidase. Polysubstance users are also found to be using synthetic cathinone and cathine concurrently with alcohol leading to haemorrhage and intoxication. Moreover, khat use have been found to hinder antipsychotic and tuberculosis medication effects on patients who are khat users. With the widespread global use of khat together with the co-administration with clinically used drugs, the detrimental effects of khat-drug interactions are pushed towards the centre of the attention. According to the National Drug Intelligence Center, a component of the U.S. Department of Justice, the potency of harvested khat fades after 48hours as cathinone degrades into cathine (https://www.justice.gov/archive/ndic/pubs31/31482/index.htm). Thus, the effects of cathine on human drug metabolizing enzymes and the possible herb-drug interactions caused by cathine warrant further exploration.It was found that a majority of khat users used more than one other psychoactive substances in which cigarettes were mainly used to maximize the stimulation power of khat with a combination of alcohol to break the aftereffect. CYPs are predominantly expressed in the liver, besides also occurring in the small intestines, lungs, placenta and kidneys. In mammals, CYP family 1, 2, 3 and 4 are involved in detoxification, steroid and eicosanoids metabolism. In humans, CYP family 1, 2, 3 and 4 and to a lesser extent CYP family 5, 8, 19, 21 and 26 are involved in xenobiotic metabolism. The CYP enzymes are predisposed to inhibition or induction by xenobiotics including herbal medications that comprise mixtures of phytochemicals. Phytochemicals such as resveratrol and quercetin have been reported to significantly inhibit CYP3A4 which translates to reduced clearance and subsequent toxicity from other CYP3A4 substrates in humans.23 There are few well characterized examples including resveratrol and quercetin which were classified as clinically significant perpetrators as numerous herbal preparations interact with CYPs in vitro but showed variable in vivo interactions.Cytochrome P450 (CYP) are a crucial family of enzymes for producing cholesterol, steroids, prostacyclins and thromboxane A2 besides playing an essential role in detoxification of foreign substances and drugs.in vitro, suggesting that other active constituents in the khat plant such as merucathine, merucathinone, tannins, cathedulins and norephedrine were likely responsible for these inhibitions. Ensuing this study, it was found that KEE also inhibited other major human drug metabolizing CYP isoforms namely CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 except CYP1A2 in vitro. Our ongoing studies, hence, are investigating modulatory effects of main active compounds of khat on the major drug metabolizing CYPs in vitro. The current study aimed to follow up on the in vitro inhibitory effects and mode of inhibitions of cathine, which is the second main active component present in khat plant, on several major drug metabolizing CYPs namely CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5. Additionally, molecular docking was performed to understand how cathine interacts with particular CYP isoforms for which its in vitro results showed significant inhibition.Investigations into the inhibitory effects of cathine on CYPs are generally lacking in the literature as most studies about khat-drug interactions have focused on cathinone, which is deemed to be the major active compound in khat. Our earlier study found that khat ethanol extract (KEE) inhibited CYP2C9, CYP2D6 and CYP3A4 significantly, but cathinone showed negligible inhibition on these CYP isoforms 1 S, 2 S)-2-amino-1-phenylpropan-1-ol.hydrochloride) was acquired from Lipomed AG . Acetonitrile was obtained from Fisher Scientific . The Vivid® CYP450 Screening kits for all CYP isoforms namely CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5 were obtained from Life Technologies™ . Costar Black 96-well plates and tris-base powder were obtained from Thermo Fisher Scientific  and Amresco® LLC , respectively.Cathine or d-Cathine.HCl  produced by blue (3-cyano-7-hydroxycoumarin), green (fluorescein) and cyan (7-hydroxy-4-trifluoromethylcoumarin) standards against a range of concentrations of the respective standard was plotted to derive the standard curves. The standard curve equation was used in succeeding assays to enumerate the fluorescent metabolites produced. Time curves were plotted afterwards to define the incubation time for single CYP assays namely 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5, respectively, as determined previously.® CYP450 Screening Kits for all CYP isoforms including Vivid® EOMCC CYP1A2 Blue, Vivid® CC CYP2A6 Blue, Vivid® BOMCC CYP2B6 Blue, Vivid® DBOMF CYP2C8 Green, Vivid® BOMCC CYP2C9 BLUE Vivid® EOMCC CYP2C19 Blue, Vivid® EOMCC CYP2D6 BLUE Vivid® EOMCC CYP2E1 Blue, Vivid® MOBFC CYP2J2 Cyan Vivid® BOMCC CYP3A4 BLUE and Vivid® BOMCC CYP3A5 Blue, according to manufacturer’s instructions. Using the 96-well black plates, in each well, 40 μL of respective reaction buffers  was added with 50 μL of master premix  and 0.3 μ/mL glucose-6-phosphate dehydrogenase in 100 mM potassium phosphate at pH 8.0) for incubation with shaking at room temperature for 30 minutes. 10 μL per well of a mixture of respective substrates and NADP+ were added to kick start the reaction. Total volume per well was 100 μL in the 96-well plate.The inhibitory effects of cathine on human CYP enzymes activities were determined using Vivid® BOMCC (7-benzyloxymethyloxy-3-cyanocoumarin) for CYP2B6, CYP2C9, CYP3A4 and CYP3A5; Vivid® CC (3-cyanocoumarin) for CYP2A6; Vivid® DBOMF (dibenzylmethylfluorescein) for CYP2C8; Vivid® EOMCC (ethoxymethyloxy-3-cyanocoumarin) for CYP1A2, CYP2C19, CYP2D6 and CYP2E1; Vivid® MOBFC (7-p-methoxy-benzyloxy-4-trifluorocoumarin) for CYP2J2; and 0.03 mM NADP+. The mixture was shaken at room temperature, 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and 120 minutes for CYP3A5. 50 μL of 0.5 M tris-base solution was added lastly to halt the reaction. The enzyme activities were measured by using the Varioskan® Fluorescence Spectrophotometer  at excitation/emission wavelengths of 415/460 nm , 415/520 nm (cyan – CYP2J2) and 490/520 nm (green – CYP2C8).The CYP enzyme specific substrates added were as follows: Vivid50 values which were less than 100 μM, and then their Ki values were further assessed. Various concentrations of cathine  were incubated with Vivid® Fluorogenic Probe Substrates CC  for CYP2A6 and BOMCC  for CYP3A4.Cathine was dissolved in water to attain the 2.5 mM of cathine stock. The assay conditions were as described above. The assay conditions were as described above, with the exception that 40 μL of buffer was substituted using 40 μL of cathine with concentrations ranging from 0 to 1000 μM, which was obtained following two times serial dilution of the cathine stock. Cathine inhibited CYP2A6 and CYP3A4 according to their IC+ to produce NADPH during the pre-incubation. The mixture  was incubated for 30 minutes with shaking under similar range of concentrations of cathine as mentioned above for IC50 determination. Substrates were subsequently added to initiate the reaction, and fluorescence readings were taken after 60 minutes for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP2J2, CYP3A4 and 120 minutes for CYP3A5 incubation. The IC50 shift was derived by calculating ratios of IC50 obtained from pre-incubation with and without NADPH. IC50 shift ratio >2 implies irreversible time-dependent inhibition.In mechanism-based or time-dependent inhibition, the master premix was added with NADP50 values, the remaining enzyme activity of each cathine concentration was divided by the solvent control well (without cathine but replaced by water) and multiplied the value with 100% to obtain the percent control activity (%) for each cathine concentration. The resulting percent control activity (%) were plotted against different concentrations of cathine to attain the half maximal inhibitory concentrations (IC50) curve. IC50 values of reversible and irreversible inhibition were determined by non-linear regression analysis using GraphPad Prism 9 for Windows . Data for inhibition constant (Ki) analysis and mode of inhibition were evaluated using Excel spreadsheet  and Lineweaver-Burk plots. The secondary plots of cathine concentrations against slopes of Lineweaver-Burk plots were plotted to determine the Ki values. All assays were carried out in triplicate and stated as mean ± SD.To determine IC Cathine conformations were generated using Open Babel GUI v2.3.1. The protein was prepared using AutoDock Tools 1.5.6 while AutoDock 4.2 software was used to carry out molecular docking. AutoGrid was used to create the grid box around co-crystallized ligand covering the active site of CYPs, which included the haem group. The grid size for specifying the search space was set at 60 × 60 × 60 Å (for CYP3A4) and 70 × 70 × 70 Å (for CYP2A6) with a default grid point spacing of 0.375 Å with a total of 10 docking runs. Docking simulations were performed using the Lamarckian genetic algorithm with 25 × 105 energy evaluations and 27 000 iterations per run. The best configuration and possible ligand binding was then determined based on the binding score. After docking, analysis of the ligand interactions at the binding site was performed and visualized using the PyMOL Molecular Graphics system .The crystal structures of human cytochrome P450 CYP2A6 (PDB code: 2FDV) and CYP3A4 (PDB code: 4D75) were retrieved from Protein Data Bank.R2 values are obtained: (i) blue (3-cyano-7-hydroxycoumarin) standard with Buffer I, II and III, y = 0.0083x + 0.012, R2 = 0.9995, y = 0.0063x + 0.0132, R2 = 0.9983, y = 0.0093x + 0.0149, R2 = 0.998, respectively, (ii) cyan (7-hydroxy-4-trifluoromethylcoumarin) standard with Buffer II, y = 0.0003x + 0.0005, R2 = 0.9811 and (iii) green (fluorescein) standard to obtain the standard equations and R2 values, y = 0.0051x + 0.0029, R2 = 0.9978. Linear ranges of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1 and CYP2J2 were from 0 – 60 minutes except 0 – 120 minutes for CYP3A5, and therefore, the incubation time was 1 hour for all CYP isoforms mentioned except 2 hours for CYP3A5 as determined in previous studies.Standard curves were plotted using fluorescence readings (RFU) against standard concentrations, and the following standard equations and 50 values of 80 and 90 μM as shown in non-linear graphs . A study by Bedada et al using human volunteers on one week of daily khat use (400 g) showed khat significantly inhibited CYP2D6, marginally inhibited CYP3A4, CYP2C19 and CYP1A2. An in vivo study using rats found that rats treated with both khat plus clopidogrel had significantly lower clopidogrel metabolite produced due to inhibition of CYP enzyme by khat. These studies supported that khat inhibited CYP activities but did not make any investigations on the specific compounds in khat that contributed to the CYPs inhibition. Therefore, it is plain to see that ingredients other than cathinone such as cathine and norephedrine, for instance, could be involved in the CYP inhibitory effects of khat.With regard to herb-drug interaction via CYP inhibition by khat constituents, our previous study found that khat significantly inhibited CYP2D6, CYP2C9 and CYP3A4 while cathinone showed negligible inhibitory effects.50 values  inhibition on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5 . Cathine0 values  of 80 anurk plot . Non-comin vitro study, cathine, the compound which cathinone decomposes into after khat is harvested, appears to only significantly inhibit CYP2A6 and CYP3A4 out of the 11 CYP isoforms tested. CYP2A6 and CYP3A4 are abundantly expressed in the liver, with an expression rate of 3.8% and 12.0%, respectively. These CYPs played a major role in metabolizing various drugs. CYP2A6 metabolizes nicotine (the primary psychoactive component in cigarettes), cotinine, tegafur, letrozole, coumarin, methoxyflurane, efavirenz, valproic acid, pilocarpine, artemisinin, artesunate, caffeine, disulfiram, halothane, fadrozole and tyrosol,41 whereas CYP3A4 is responsible for 40% to 45% of all phase I metabolism and approximately 70% of gastrointestinal CYP activity beside being co-expressed with P-glycoprotein (PGP) in the liver and intestines.43 CYP3A4 metabolizes drugs such as paclitaxel, fentanyl, tamoxifen, tacrolimus and statins. Therefore, khat/cathine users who are on medications metabolized by CYP2A6 and CYP3A4 may experience herb-drug interactions or may fail to respond to some medications. Further human studies are warranted to investigate whether the inhibitory effects of khat/cathine on CYPs are clinically significant or otherwise.Based on our  The average content of cathine in 100 g of fresh khat leaves is 83-120 mg10. A 100-400 g of fresh khat leaves is consumed on a daily basis. Hence, assuming that khat users who took 400 g of fresh khat leaves would have consumed about 332-480 mg of cathine, therefore, an average 70 kg adult with 5 L blood would have absorbed approximately 66.4 × 10−3 to 96 ×10−3 mg/ml of cathine or in other words, about 351.7-511.5 μM. Despite the low Ki values determined in our study that ranges from 63 to 100 μM, these values may be clinically relevant since khat users commonly take huge amount of khat on a daily basis. Toennes et al reported that at a mean of 43.8 g of khat chewed, the mean ingested dose of cathine in milligrams was 32.4, while the absorbed proportion of cathine ranges from 16% to 84%. The mucosa of the oral cavity is the first absorption segment, where the major fraction of the cathine (84 ± 6%) is absorbed. Cathine could be detected in the urine up to 50-70 hours after ingestion which was about a day time more than cathinone at 22-26 hours. In addition, a study found that cathine concentration in oral fluid was higher than that of cathinone because khat users did not chew fresh leaves and cathinone easily metabolized into cathine. According to European Monitoring Centre for Drugs and Drug Addiction, hepatic first-pass metabolism of cathinone forms norephedrine and only 2% of cathinone is excreted in the urine (https://www.emcdda.europa.eu/publications/drug-profiles/khat). The elimination half-life of cathine (5.2 ± 3.4 hours) is longer than cathinone (1.5 ± 0.8 hours) (https://www.emcdda.europa.eu/publications/drug-profiles/khat). As compared to cathinone, cathine is less lipophilic and may also penetrate to the central nervous system. Nevertheless, lipophilicity of cathine should also be considered during prescribing by healthcare professionals to khat users because lipophilicity impacts cellular uptake and ADMET . High dosage accompanied by high lipophilicity compound is a detrimental combination. Although less lipophilic than cathinone, the lipophilicity of cathine may still affect metabolic activity as lipophilic compounds showed greater affinity to metabolic enzymes. Khat compounds’ lipophilicity need to be known during drug administration as they may readily cross the blood brain barrier.To our best knowledge, pharmacokinetic studies on cathine are very limited. Khat chewing releases cathine and cathinone which was subsequently absorbed by the mucous membranes of the oral cavity and followed by the stomach lining. Dried khat leaves showed an increase in cathine amount from 0.172% to 0.192% (fresh khat) to 0.184-0.198%. According to one case report, a male patient who suffered from haemorrhagic stroke with hypertension was found to have no chronic diseases history but instead daily khat chewing. The patient’s urine samples showed positive for amphetamine-like substance while liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected presence of cathine and cathinone. The serum concentration of cathine was 100-fold of cathinone, suggesting that the patient’s symptoms are most likely due to cathine. The maximal plasma concentrations of cathine will be reached at 2.6 hours with a mean residence time (MRT) of 10.2 ± 2.6 hours, which was twice more than cathinone. Cathine retain in the human body for a longer period of time as compared to cathinone and could be detected in the urine samples even after approximately ≥3 days after ingestion. Since cathine is retained for a longer period of time within the human body, it may exert greater inhibitory effects on human CYPs and higher chances of causing khat-drug interactions as compared to cathinone. Nevertheless, the effects of cathine retaining within the body for a longer time have yet to be explored.However, in the early 1945-1961, there were contradicting debates by researchers that cathine has low potency and the amount of cathine in khat is insufficient to exert symptoms shown after consumption versus cathine was the only khat alkaloid responsible to give pharmacological significance.in vitro findings showed non-competitive mixed mode of inhibition, our molecular docking analysis provided a prediction of the interaction of cathine with the active site of CYP2A6 and CYP3A4 which may contribute to the mixed mode inhibition component. Intermolecular interactions namely hydrogen bonding and hydrophobic interactions were also previously established to be the chief determinants in determining the stability of energetically favoured ligands within the binding pocket. Cathine interacted with CYP2A6 via four hydrophobic interactions with Ile300, Thr305, Phe480, hydrogen bonding with Gly301 and Thr305 and single π-stacking with Phe107 and Phe118  more strongly than CYP3A4 (Ki = 100 μM). This conclusion is warranted by the binding energy value of the CYP2A6-cathine complex of −7.96 kcal mol−1 that exceeds the binding energy value of CYP3A4-cathine complex of −6.53 kcal mol−1. Non-polar or hydrophobic interactions are known to play a major role in contributing to binding forces and folding stability of the proteins or in other words the CYP enzyme while charged residues interacting with polar groups in the form of hydrogen bonds reinforce specificity. Both cathine-CYP complexes showed similar hydrogen bonds on one end of the cathine molecule . In CYP2A6, cathine overlaps very well with aldrithiol while ketoconazole is much bigger in size and has more interactions as compared to cathine in CYP3A4. In the active site of CYP2A6, aldrithiol formed hydrogen bonds with Asn297, π-stacking with Phe107, Phe111, and Phe118 and hydrophobic bonds with Phe209, Ile300, Gly301, Thr305 and Phe408. Ketoconazole formed π-stacking with Phe304, and strong ionic interaction with Fe2+ of the haem group of CYP3A4 and hydrophobic bonds with Phe57, Arg105, Ser119, Leu210, Phe241, Ile301, Ala305, Ala370, Arg372, Glu374, Gly481 and Leu482. Some similar interactions seen from our docking outcome such as (1) in CYP2A6, cathine and aldrithiol  both have a series of π-stacking interactions with Phe107, Phe118 and Phe480 to stabilize them, hydrophobic interactions with Thr305 and (2) in CYP3A4, cathine and ketoconazole both have π-stacking with Phe304 formed.We have performed docking using known inhibitors of CYP2A6 (PDB 2FDY) – aldrithiol and CYP3A4 (PDB 2V0M) – ketoconazole from Protein Data Bank to compare with cathine’s interactions. Cathine overlaps well with experimentally determined binding modes of aldrithiol and ketoconazole  of the substrate (e.g. cathine). Further studies should employ docking combined with molecular dynamics simulations to investigate effects of SOM flexibility of CYPs. The in vitro mixed mode inhibition results have raised another limitation of this study especially when mixed mode of inhibition suggests multiple possible binding modes but could not be predicted from our docking study. What is more, the allosteric sites of CYP enzymes are an area of active research, although locations among helices C, E and H have recently been proposed.The current study utilizes high-throughput fluorescence-based assays for detection of enzyme-drug interactions.in vitro and docking predictions are lacking in biotransformation capabilities, and the lack of in vivo biological mechanisms has probably stronger impact on the accuracy of toxicity evaluations as cathine inhibitory effects on CYPs were performed in vitro. This study answers some previous questions regarding the inhibitory effects of cathine on major human drug metabolizing CYP enzymes but also raises new questions to explore. As mentioned earlier that cathine is retained in the human body for a longer period of time as compared to cathinone, therefore future studies should consider effects of cathine on CYP1A1 that are present in large amount in gut cells, shedding light to enteric drug interaction potential. Research has also shown that (1R)(2S)-norephedrine was not the final product produced from the phenypropylamino alkaloids pathway in khat plant as claimed in earlier studies, but instead the metabolic pathway continues downstream of cathine to form oxazolidine derivates. Therefore, future studies are recommended to look into the inhibitory effects in vitro and in silico of other khat constituents including norephedrine and oxazolidine derivatives. This area of research remains widely unexplored, giving room for further research on the unique phytochemistry of khat to better understand the khat-drug interactions besides detecting possible similar phytoconstituents that are also present in other herbal products, thus indirectly helping in understanding therapeutic potential of other herbs as well. Future in vivo studies using human subjects and animal models are also warranted to look into the effect of specific khat contents, for instance, cathinone, cathine, norephedrine, merucathine, merucathinone, cathedulins and tannins.It is worth mentioning that in vitro inhibition determined as cathine inhibited CYP2A6 with Ki value of 63 μM which was stronger than CYP3A4 with Ki value of 100 μM. This deduction is justified by the binding energy value of the CYP2A6-cathine complex of −7.96 kcal mol−1 that exceeds the binding energy value of CYP3A4-cathine complex of −6.53 kcal mol−1. Additional π-stacking with nearby phenylalanine residues in CYP2A6 may explain why cathine showed more stable and stronger affinity on CYP2A6 than CYP3A4. In silico, in vitro and in vivo research on cathine are scarcely explored, and this study offers useful insights into the molecular interaction of cathine with CYPs besides directing future studies in the khat-drug interaction area. This study is a preliminary screening that must be validated by means of various approaches. Nevertheless, in view of the budding khat and cathinone derivatives’ use, knowledge on khat-drug, cathinone-drug or cathine-drug interactions are urgently needed. The current in vitro preliminary findings of this study require more diverse models  before a comprehensive exploration in clinical trials. The current study showed synergistic results from both in vitro and in silico docking evaluation of cathine as the inhibitor of CYPs enzyme activities. Our study proved that cathine exhibited negligible in vitro inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2 and CYP3A5, and significant inhibition on CYP2A6 and CYP3A4. Despite this preliminary in vitro screening outcome, it would be best to recommend healthcare experts and patients to not co-administer khat or cathine-containing products with clinical drugs to minimize the possible cathine-drug interactions, especially those metabolized by CYP2A6 and CYP3A4.Cathine reversibly inhibited CYP2A6 and CYP3A4 activities. Docking results of CYP2A6 and CYP3A4 is aligned to the Click here for additional data file.In Vitro Inhibitory Effects of Cathine on 11 Major Human Drug Metabolising Cytochrome P450s by Sharoen Y. M. Lim, Jason Siau Ee Loo, Mustafa Alshagga, Mohammed A. Alshawsh, Chin E. Ong, and Yan Pan in International Journal of ToxicologySupplemental Material for Protein-Ligand Identification and


Solanum tuberosum L.), 14-3-3 protein forms a protein complex with the FLOWERING LOCUS T (FT)-like protein StSP6A and the FD-like protein StFDL1 to activate potato tuber formation. Eleven 14-3-3 isoforms were reported in potato, designated as St14a-k. In this study, the crystal structure of the free form of St14f was determined at 2.5 Å resolution. Three chains were included in the asymmetric unit of the St14f free form crystal, and the structural deviation among the three chain structures was found on the C-terminal helix H and I. The St14f free form structure in solution was also investigated by nuclear magnetic resonance (NMR) residual dipolar coupling analysis, and the chain B in the crystal structure was consistent with NMR data. Compared to other crystal structures, St14f helix I exhibited a different conformation with larger B-factor values. Larger B-factor values on helix I were also found in the 14-3-3 free form structure with higher solvent contents. The mutation in St14f Helix I stabilized the complex with StFDL1. These data clearly showed that the flexibility of helix I of 14-3-3 protein plays an important role in the recognition of target protein.In potato ( The number of 14-3-3 isoforms of animals is 7 in most cases, but the number of 14-3-3 isoforms of plants depends on the species, for example, 15 in Arabidopsis4, 12 in tomato6, 17 in tobacco7, 11 in potato8, and 8 in rice10. The 14-3-3 proteins, including all isoforms, belong to a single family with structural similarity and are thought to function as homo- or heterodimers12.The 14-3-3 proteins are ubiquitously expressed in all eukaryotic cells and are involved in the regulation of diverse cellular functions, such as metabolism, signal transduction, cell cycle control, apoptosis, transcription, and protein trafficking15. Large-scale protein interactomics mapping and mass spectrometry-based studies have revealed more than 300 proteins putatively binding 14-3-3 in plants16. In rice, the nonepsilon 14-3-3 protein GF14 is an interactor of Hd3a and OsFD1 proteins. Hd3a is a rice homolog of Arabidopsis FLOWERING LOCUS T (FT) protein, which induces flowering17. OsFD1 is the basic leucine zipper (bZIP) transcription factor and a typical 14-3-3 binding protein; it possesses a 14-3-3 binding motif with a phosphorylated Ser/Thr residue and it interacts with 14-3-3 at the binding pocket of the phosphate group of phosphorylated proteins18. In potato, the nonepsilon 14-3-3 protein St14 interacts with the potato homolog of FT protein, StSP6A, and bZIP transcription factor StFDL1 and regulates potato tuber formation8.Plant 14-3-3 proteins are divided into two groups based on their gene structures, namely, epsilon and nonepsilon, and the latter is plant-specific18. The 14-3-3 proteins have been recognized as possessing a similar and rigid conformation in both free- and complex- forms because the structures of the free- and complex-forms of both human 14-3-3 zeta and sigma were similar19. However, recently, the structural difference between free- and complex-forms of human 14-3-3 beta was reported as the open- and closed-structures, respectively20. Furthermore, molecular dynamics (MD) simulation of human 14-3-3 sigma showed structural flexibility and suggested the possibility of facilitating the binding of its ligand21.Crystal structures of 14-3-3 proteins of both animals and plants have been determined, revealing that 14-3-3 proteins form homodimers, with a characteristic cup-like or horseshoe structure possessing positively charged grooves for pSer or pThrIn this work, the free form structure of St14f was determined by X-ray crystallography at 2.5 Å resolution and compared with the other 14-3-3s. The structure of the I helix of St14f was different from others, and the importance of the I helix of St14f on StFDL1 recognition was studied by nuclear magnetic resonance (NMR) and thermal shift assays.The crystal structure of the St14f free form was determined at 2.5 Å. Three chains  are contained in the asymmetric unit, i.e., two conformations of dimers (AA and BC) exist in the crystal Figs. . The St122. St14f showed 82% amino acid sequence identity with GF14c, the structure of which was determined in complex with OsFD1 and Hd3a. The whole structure of the GF14c complex form was similar to the structure of the St14f free form, but the local structure in helices H and I was different, and the grooves were narrower than the grooves in the St14f free form . Superposition was performed using the CCP4 Superpose program, which was the structural alignment of the C-alpha backbone based on secondary structure matchingrm Figs. . The frerm Figs. . The grorm Figs. . In contrm Figs. . Helicesrm Figs. .Figure 215N Lys-labeled St14f were measured with and without StFDL1 peptide (LNRTSpTAPF). Assignment of Lys residues was carried out using point mutation for both free and St14f-StFDL1 complex forms  were found and possessed different conformations, named the A, B, and C structures (free form), respectively. To evaluate which conformation of St14f is close to the solution structure, NMR was used. Residual Dipolar Couplings (RDCs) values measured by NMR provide long-distance structural information for proteins in solution. RDC values were experimentally obtained from TROSY and HSQC spectra using Pf1 phage as an aligned medium.23  of St14f were calculated and are depicted in Fig. 2, whereas the average B-factors of helices H and I were 73 and 98 Å2, respectively, suggesting that helices H and I are more flexible than other helices. The standard deviations of helices H and I were large, 26 Å2 (36%) and 24 Å2 (25%), respectively, suggesting that helices H and I contain both flexible and nonflexible residues. NMR experiments, such as 1H-15N hetero-nuclear Overhauser effect (NOE), were performed to evaluate the flexibility in solution, but they did not work well because of the lower sensitivity of NMR signals for the St14f homodimer with a larger molecular weight of ~ 60 kDa.The B-factor of an atom is a potential measure to evaluate the flexibility because the B-factor is determined by its intrinsic flexibility and possible crystal packing effects20. This structural difference suggests that the 14-3-3 protein is flexible. The MD simulation study of human 14-3-3 sigma suggests the presence of large-amplitude motion within the 14-3-3 molecule, i.e., helices A-D and G-I are rigid, but helices E and F are flexible21. In this study, we found that helix I is flexible, which has not yet been reported. There are some reasons why we could find this flexibility. First, our crystal structure is determined for the 14-3-3 free form. Most 14-3-3 structures are complex forms where the flexibility of helix I is not expected. Second, our crystal structure possesses the appropriate B-factor values to analyze the flexibility of helix I by chance. This flexibility is difficult to find because B-factors at medium resolution structures tend to be larger and less reliable than at higher resolution structures, and only 2 of 9 crystal structures of the 14-3-3 free form show clear differences  were analyzed  was calculated with and without the StFDL1 peptide Table , Fig. 4e. discussed that helix I may promote the correct conformation and location of the phosphorylated residue of the binding partner in the major binding groove of 14-3-3 zeta, consistent with our data showing the importance of helix I28. The presence of the flexibility of the binding partner has been discussed by Tugaeva et al.29, consistent with the discussion by Masone et al.28 It is noted that the helix I is located on the binding surface of B-Raf30, Nth131, pHSPB632, and Hd3a17, and plays an important role in these client protein bindings. Moreover, D228V mutation on helix I of human 14-3-3 gamma is related to the disease, developmental and epileptic encephalopathy 5633. The flexibility of helix I and/or the binding partners may allow the recognition of so many kinds of binding partners.We also found the importance of helix I in the recognition of the binding partner. Masone et al34. The construct was transformed into E. coli Rosetta (DE3). Cells were grown at 310 K in lysogeny broth medium containing 0.1 mg/mL ampicillin. Overexpression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM at an optical density (OD) at 600 nm of 0.5–0.6. Cells were incubated at 288 K for 16 h under shaking at 100 rpm, harvested by centrifugation, and suspended in buffer . The cell suspension was sonicated and centrifuged. The supernatant was loaded onto a Glutathione Sepharose 4B (GS4B)  column equilibrated with the same buffer . GS4B resin was washed with the same buffer, and then the protein was eluted with a buffer . The elution was mixed with HRV 3C protease and dialyzed against 1 L of buffer  at 277 K for 12 h for cleavage. The protein was rechromatographed using the GS4B affinity column to remove the residual affinity tag. The target protein was subjected to size exclusion chromatography equilibrated with buffer .The pCold GST vector contains a DNA fragment corresponding to St14fThe purified St14f was dialyzed against 1 L of buffer  and concentrated to 10 mg/mL (0.34 mM). The protein concentrations were determined using ultraviolet (UV) absorption measurements at 280 nm. The hanging drop vapor diffusion method is used for crystallization. Drops were made of 1 µL of protein and 1 µL of reservoir solution and suspended over 0.5 mL of reservoir solution at 277 K. St14f formed crystals of approximately 0.2 mm × 0.2 mm × 0.2 mm in dimension within 3 days using a reservoir solution of 12% (w/v) polyethylene glycol (PEG) 8000, 0.2 M ammonium tartrate pH 7. The crystal was sequentially transferred to 20% (v/v) ethylene glycol in the reservoir buffer using 2% increments and flash cooled in liquid nitrogen.35. Phasing of St14f was performed by molecular replacement with 14-3-3 protein from Nicotiana tabacum  using Phaser36. Structure refinement was performed with COOT37 and REFMAC538 using the TLS refinement strategy40. TLS method extracts a linear combination of anisotropic translational (T), librational (L), and screw (S) motions from an analysis of the isotropic temperature factors of the atomic coordinates refined against X-ray diffraction data. The rigid-body motions of the peptide segments of potato 14-3-3 protein St14f were analyzed by the TLS method using the coordinates for St14f free form where the 233-residue amino acid sequence was partitioned into 20 TLS groups using the web-based server, http://skuld.bmsc.washington.edu/~tlsmd38. The structural model was evaluated using RAMPAGE41. The coordinate and structure factors have been deposited with the PDB with accession codes 7xbq (with TLS refinement). Data collection and refinement statistics including the space group are given in Table The diffraction dataset was collected at 100 K using an MX300HE CCD detector (Rayonix) on the BL44XU beamline at SPring-8 . The crystal-to-detector distance was set to 350 mm. Two hundred oscillations of 1° were collected with an exposure time of 2 s. The dataset was obtained at a wavelength of 0.9 Å. Reflections were integrated and scaled by HKL-20001H-15N-HSQC spectrum of the St14f free (red) and StFDL1 peptide complex (blue) at 298 K using a Bruker AVANCE III 950 MHz spectrometer equipped with a 1H/13C/15N TCI CryoProbe (Bruker BioSpin).The 15N Lys-labeled St14f was prepared at 250 µM in a buffer containing 10 mM Tris, pH 7.5, 1 mM DTT, and 10% D2O. The complex sample was prepared with a final concentration of 1 mM StFDL1 peptide and 250 µM 15N Lys-labeled St14f. The spectrum was processed using the TopSpin software package (Bruker BioSpin). Assignments were carried out using single point mutations of St14f . All experiments were conducted at 298 K and 950 MHz. Alignments of the samples containing Pf1 phage were confirmed by quadrupolar splittings of D2O signals in 1D 2H spectra, which were measured in conjunction with HSQC or TROSY measurements.RDCs were extracted from the chemical shift differences and resonance splitting measured from HSQC and TROSY spectra, which were collected on 0.5 mM St14f in 50 mM potassium phosphate buffer pH 6.8, 50 mM KCl, 10% DThe St14f mutants were prepared by inverse PCR using KOD Plus (Toyobo). Purified St14f protein (10 mM HEPES pH 7.5 and 0.15 M NaCl buffer) was added to SYPRO Orange dye (stock concentration: 5000×) (Sigma). The final volume of each reaction was 20 µL, and the final concentration of St14f was 10 µM with no StFDL1 peptide and with 100 µM StFDL1 peptide. The 96-well plate was heated from 25 to 99 °C with a ramp rate of 1 continuous. The thermal shift assay was conducted in the Applied Biosystems real-time PCR instrument (API). The nonlinear fitting of thermal denaturation curves was performed to derive melting temperatures using Protein Thermal Shift Software.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.Supplementary Figure S8.Supplementary Figure S9.Supplementary Tables.


Herein, we propose a facile metal–organic framework (MOF) derived strategy to prepare Bi2O3 microrods with a carbon coat (Bi2O3@C). They exhibit ultrahigh specific capacity (1378 C g−1 at 0.5 A g−1) and excellent cycling stability (93% retention at 4000 cycles) when acting as negative electrode material for advanced asymmetric SCs. The assembled Bi2O3@C//CoNi-LDH asymmetric supercapacitor device exhibits a high energy density of 49 W h kg−1 at a power density of 807 W kg−1. The current Bi-MOF-derived strategy would provide valuable insights to prepare Bi-based inorganic nanomaterials for high-performance energy storage technologies and beyond.Bismuth oxide (Bi 2O3 microrods with a carbon coat (Bi2O3@C) exhibit ultrahigh specific capacity (1378 C g−1 at 0.5 A g−1) and excellent cycling stability (93% retention at 4000 cycles) as negative electrodes for supercapacitors.Bi Carbon-based materials are usually employed as negative electrodes with excellent rate and long lifespan. However, the low specific capacity of carbon materials still restricts the energy density of ASCs for practical applications.5,6 Therefore, it is imperative to seek low-cost, high specific capacity and durable negative electrode materials to meet the increasing requirements of peak-power electric vehicles.7Pollution and depletion of fossil fuels have caused environmental issues and an energy crisis, and it is urgent to develop environmentally friendly and efficient energy storage equipment.2O3) has been considered as a promising negative electrode material due to its cheaper, environmental friendliness, abundant resources and high theoretical specific capacities (1370 C g−1 at 1 A g−1).8,9 For instance, Qiu et al.10 synthesized ultrathin Bi2O3 nanowires by oxidative metal vapor transport deposition technique, which exhibited high specific capacity (576 C g−1 at 2 A g−1). Shinde et al.11 grew 3D Bi2O3 by fast chemical method at room temperature, which demonstrated a specific capacity of 447 C g−1 at current density of 2 A g−1. Liu et al.12 designed oxygen-deficient r-Bi2O3/graphene flexible electrode with high specific capacity of 1137 C g−1 at 1 mA cm−2. Nevertheless, Bi2O3 still has disadvantages for ASCs, such as its intrinsically poor electronic and ionic conductivities, the large volume expansion in charging–discharging process. Improved researches have illustrated that the carbon can be used as a buffer layer, which could effectively reduce the morphological change and protect the structure of electrode. The facile design and preparation strategy of Bi2O3/C composites remains a continuing research to adjust the morphological and electronic structures.13–16Recently, bismuth oxide  as sacrificial templates to derive nanocarbons or metal compounds/composites are an effective approach to obtain excellent electrode materials with high reversible capacity and cycle performance.2O3@C microrod through a facile one-step heat treatment, using Bi-based MOF (CAU-17) as both template and precursor. Benefiting from the carbon coated layer from the pyrolysis of CAU-17, it can increase the electrical conductivity and ease the volume collapse during the discharge–charge process. The obtained Bi2O3@C hybrid as negative electrode for SCs exhibits ultrahigh specific capacity (1378 C g−1 at 0.5 A g−1) and excellent cycling stability (93% retention at 4000 cycles). In addition, ASC device using a layered double hydroxide (CoNi-LDH) as positive electrode provides a high energy density of 49 W h kg−1 at a power density of 807 W kg−1.Herein for the first time, we report a Bi−1. Scanning electron microscopy (SEM) images were obtained from Hitachi S-4800 a field emission scanning electron microscope (FESEM) equipped with an energy dispersive spectrometer (EDS) and mapping operated at an acceleration voltage of 10.0 kV. Transmission electron microscope (TEM) images were recorded using a Thermo Fischer Talos F200× with an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were performed on a Thermo Scientific ESCALAB250-Xi. Thermogravimetric analysis (TG) curve was measured on SII TG/DTA 7300 instrument at a heating rate of 10 °C min−1 under N2 atmosphere.Powder X-ray diffraction (PXRD) patterns were implemented by a Bruker AXS D8 Advance diffractometer at 40 kV, 40 mA using a Cu Kα (1.5406 Å) at room temperature. The Fourier transformation infrared spectra (FTIR) were carried out on a NICOLET-6700 infrared spectrometer using the KBr pellet method in the range of 400–4000 cm3)3·5H2O were ground uniformly and added to 60 mL methanol. Putting the mixture dissolved under ultrasonic for 10 min. Then homogeneous solution transferring to a Teflon-lined stainless-steel autoclave and heated at 120 °C for 24 h. After cooling down to room temperature, the white powder were collected by filtration and washed with methanol. The final samples were dried at 60 °C for 3 h.All reagents are commercially produced without further purification. Typically, 715 mg of 1,3,5-benzenetricarboxylic acid and 150 mg of Bi2·6H2O, 2 mmol Ni(NO3)2·6H2O and 8 mmol hexamethylenetetramine (HMT) were dissolved in 30 mL distilled water under magnetic stirring to form solution, then the solution was transferred into 50 mL autoclave. The autoclave was sealed and maintained at 100 °C for 10 h, and then cooled naturally. Finally, the products were washed with H2O and ethanol, and then dried at 60 °C for 6 h.Typically, 2 mmol Co(NO2O3@C samples (80 wt%), acetylene black (10 wt%), and polyvinylidene fluoride (PVDF) (10 wt%) with the 1-methyl-2-pyrrolidinone (NMP) to form a slurry. Then, the slurry was coated onto the nickel foam substrates (1.0 cm × 1.0 cm), and dried at 60 °C for over-night, after pressed at 10 MPa for approximately one minute. Cyclic voltammetry (CV), galvanostatic charge–discharge (GCD) and electrochemical impedance spectroscopy (EIS) were used to evaluate the electrochemical performances of the working electrode.The electrochemical performance of the tested electrodes was evaluated in a three-electrode system where a saturated calomel electrode (SCE) as the reference electrode and Pt wire counter electrode, respectively, which in alkaline aqueous solution by an electrochemical analyzer system, CHI660E . The working electrodes were fabricated from a mixture containing the BiQs, C g−1) were calculated by the following equation:I is the current (A), Δt is the discharge time (s), m is the mass of electrode active materials (g).The specific capacity (q+ = q−. The energy density (E) and power density (P) are calculated based on the total mass of the active materials of the two electrodes, according to the following equations:V is the voltage window, Δt is the full discharge time of the assembled ASC devices.Before assembling an asymmetric supercapacitor, the load mass of the two working electrodes is balanced by the relationship of charge via a solvothermal method according to our previous report.20 XRD pattern of the precursor matched well with the phase purity of CAU-17 .23 Consequently, Bi2O3@C was obtained by thermal treatment in nitrogen atmosphere and the heating temperature was optimized to be 500 °C (ESIθ correspond to (012), (022) and (221) planes, respectively, which can be well indexed to monoclinic α-Bi2O3 phase (JCPDS card no. 71-0465).24 The EDS spectrum  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, and O–CO, respectively.9XPS test is further carried out to analyse the chemical compositions and detailed surface electronic states. The survey spectrum displays that the existence of Bi, C and O elements in as-prepared materials , which a profile  indicate2O3@C shows that the microrod morphology remains intact after the pyrolysis, while the hexagonal configuration is damaged. The smooth surface indicates that Bi2O3 is well encapsulated within the carbon layer. Benefiting from the periodic arrangement of organic motifs and metal nodes in Bi-MOF structure, the carbon formed in situ is uniformly coated on the surface of Bi2O3. Such unique hybrid structure may improve the conductivity and stability, which are beneficial for energy storage. The average diameter and length of Bi2O3@C microrods are about 820 nm and 3 μm, respectively, which are smaller than CAU-17 precursor (∼1.0 μm and ∼3.5 μm). This size shrinkage could be associated with the massive loss of organic components during the annealing process.27 TEM result  plane of Bi2O3. Additionally, SAED pattern present a good crystallinity of Bi2O3. Elemental mappings of an individual Bi2O3@C microrod indicate all elements are uniformly distributed in the whole structure  are revealed in 2O3@C electrode exhibits remarkable specific capacity, which are 1378, 1095, 937, 818 and 575 C g−1 at 0.5, 1, 2, 3 and 5 A g−1 respectively. The considerable specific capacity is superior to some previously related materials, such as AC (activated carbon)–Bi2O3 electrode (333 C g−1 at 1 A g−1),28 CQD (carbon quantum dot)–Bi2O3 (343 C g−1 at 0.5 A g−1),29 mesoporous 3-D Bi2O3 (447 C g−1 at 2 A g−1),11 Bi2O3 nanowires (576 C g−1 at 2 A g−1),10 and so on , v is the scan rate (V s−1), a is constant and b is the Power law exponent. The value of b is calculated from the slope of log i vs. log ν at a constant potential of redox, where b = 1 indicates capacitive-controlled mechanism and b = 0.5 is characteristic of diffusion-controlled mechanism.31 The obtained b value of Bi2O3@C is 0.53 indicates the diffusion charge storage is dominated. The fraction of diffusive charge storage fd is determined using the formula: fd = (1 − b)/0.5, which is used to calculate the capacitive and diffusive contributions of current.25 The value of diffusive charge storage is 0.94 for Bi2O3@C, further demonstrating the diffusion-controlled contribution. There is also a part of the capacitive-controlled contribution, which is caused by the carbon in hybrid Bi2O3@C. The EIS technique is also employed at the open-circuit potential in the frequency range from 100 kHz to 0.01 Hz. The Nyquist plots of Bi2O3@C electrode in Rs) is equal to the intercept on the X-axis (1.68 Ω), the charge-transfer resistance (Rct) corresponds to the diameter of the semicircular loop at the high frequency (0.22 Ω). Moreover, the line with large slope is named Warburg resistance in the low frequency region, suggesting Bi2O3@C has excellent ion diffusion and fast charge transport speed.In addition, the charge storage mechanism for Bi2O3@C electrode, an asymmetric supercapacitor (ASC) device is assembled in 1 M KOH electrolyte by employing Bi2O3@C/NF (nickel foam) and layered double hydroxide (CoNi-LDH/NF) as negative and positive electrode, respectively. The CoNi-LDH nanoflowers composed of ultrathin nanosheets are prepared through a typical method.20,32 The SEM and electrochemical properties of CoNi-LDH are displayed in Fig. S3.−1 at 1, 2, 3, 5 and 8 A g−1. The CV curves of ASC device are shown in −1) at the voltage range of 0–1.6 V, suggesting fast charge–discharge properties of the ASC device. The GCD curves of ASC device are shown in −1 at the current density of 1 A g−1. Ragone plots (energy density vs. power density) of ASC device is depicted in 2O3@C//CoNi-LDH device is 49 W h kg−1 at a power density of 807 W kg−1, and still maintains 9 W h kg−1 at a high power density of 4050 W kg−1, which are advantageous over many previously reported ASC, such as AC//Bi2O3@C ,33 ESCNF@Bi2O3//CF@NiCo2O4 ,34 Bi2O3//MnO2 ,35 Bi2O3–Ni–F//graphite 36 CoNi-LDH//AC .37 The capacity retention of Bi2O3@C//CoNi-LDH ASC is shown in −1, suggesting a favorable stability of this ASC device. These results confirm that as-prepared Bi2O3@C is expected to be a potential candidate as negative electrode material and satisfy requirements of high-performance ASC device.To further investigate the practical application of hybrid Bi2O3@C negative electrode material through Bi-MOF-template-directed strategy. Benefiting from the calcined MOF at N2 atmosphere, a large amount of carbon is retained in the composite product, increasing the electrical conductivity of Bi2O3@C composite materials. The Bi2O3@C as a battery-type negative electrode for SCs exhibits ultrahigh specific capacity of 1378 C g−1 at a current density of 0.5 A g−1 and excellent cycling stability of 93% retention after 4000 cycles. Moreover, a constructed Bi2O3@C//CoNi-LDH ASC device exhibits high energy density of 49 W h kg−1 at a power density of 807 W kg−1. All above-mentioned advantages demonstrate that the current approach of Bi-MOFs-derived strategy would provide valuable insights to prepare Bi-based inorganic nanomaterials for high-performance energy storage technologies and beyond.In summary, a facile one-step pyrolysis method is used to synthesize BiThere are no conflicts to declare.RA-010-D0RA01470B-s001