Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors. Furin1313via recognition at several subsites at the catalytic domain, typically involving multiple tight contacts and hydrogen bonds. The P-domain is hereby closely associated with the catalytic domain and is essential for its stabilisation, but it does not seem to be involved in the ultimate subsite recognition. Antibodies can act as highly specific protease inhibitorsH) domains; as isolated proteins often called VHH-fragments or “nanobodies”) as well as antigen binding fragments (Fabs) have been successfully applied to effectively inhibit pharmacological targets like human growth factor activator (HGFA)Crystal structures of inhibitor-bound murine111613To unravel the binding epitope and the mode-of-action of the furin-inhibiting nanobody Nb14 we solved its structure in isolation as well as in complex with the target protease. Our structural and biochemical data explain its unusual inhibitory properties and suggest strategies for the future of inhibitor development.3 were separated from these needle clusters and were used for single crystal diffraction data collection at the micro-focus beamline P14 at the European Molecular Biology Laboratory (EMBL) in Hamburg  and the inhibitory camelid antibody Nb14. Even optimised crystals grew as needle clusters. Using a whole needle-cluster, initial diffraction data were obtained to a limiting resolution of 2.9 Å at the synchrotron beamline BL14.1 of the Helmholtz-Zentrum Berlin (HZB) . These d Hamburg . The lat Hamburg .2. The interaction interface (~700 A2) is somewhat smaller than for the HGFA-inhibiting Fabs Ab58 (~900 A2) and Ab75 (~1000 A2). However, it corresponds well to the average size observed for protein-antigen complexes (varying from ~400 A2-~1000 A2 2) and basic pancreatic trypsin inhibitor iG-P) and complexation significance score (CSS) values of 0.199 and 0.293 of this interface underline the significance of the observed interaction. All other molecular interactions, either mediated by non-crystallographic or crystallographic contacts, are classified as non-significant. An unusually high number of tyrosine side chains is found in the interaction interface of furin and Nb14, including Tyr32, Tyr35, Tyr109 of Nb14 and Tyr560 of furin. These tyrosines form specific hydrogen bonds and contribute to the hydrophobic character of the surface. Analysis with the PISA serveriG) of −28.5 kJ/mol, indicating a highly hydrophobic interface.Nb14 was bound to the P-domain of furin rather than to the catalytic domain  and S2, WT) and mutated human furin (FurinT562R), respectively.To analyse the impact of the observed molecular contact, a point mutation was introduced to furin changing Thr562 into arginine. Thr562 is located at a central position of the interaction interface inset in  SubstituWT and FurinT562R in analytical gel permeation chromatography (GPC) experiments. If an equimolar mixture of FurinWT and Nb14 is subjected to gel permeation chromatography, the proteins form the expected complex and co-elute in a single peak , showing largely reduced self-degradation compared to the wild type proteaseWT and FurinT562R essentially showed the same activity in the factor XS195A cleavage assay  of 0.32 Å. Furin is found in its catalytically active state forming a typical covalent complex with the chloromethylketone compound  and the decanoylamide-C9 of the inhibitor measures approx. 19 Å. Canonical binding of peptides up to the S5 or S6 pockets is thus completely unperturbed by Nb14 and the placement of an elongated peptide chain to this region is sterically not hindered. In addition, the catalytic cleft and the active site of furin are clearly not blocked by the binding of Nb14, and its inactivity towards the cleavage of small peptidic substratesGlobular folded parts of substrate proteins do however show an additional level of access control to the active site of proteases, as they are largely sterically restricted from the substrate binding cleft. Therefore, the binding of proteinaceous substrates to furin and their cleavage should only be possible if their overall conformation fits to the specific topology of the furin:Nb14 complex. This hypothesis was tested by means of modelling studies with the coagulation factor X  and S4. We have investigated the structural basis of inhibition of human furin by the highly specific inhibitory camelid antibody Nb14The mode of action observed for Nb14 is largely different to other nanobodies that inhibit proteases by either blocking the active site cleft . Molecular tools are available (e.g. phage displayS195A The expression of camelid antibody Nb142) was crystallised in 0.1 M sodium acetate, pH 5.6, 16–18% PEG 3350. Crystals of isolated Nb14 were grown using a controlled dry-out procedure. Crystallisation drops (200 nl) of protein solution  were pipetted without any reservoir solution. The plates were sealed and crystals appeared after several days and were stable for several weeks. Diffraction data were collected at BL 14.1 of BESSY-II, Helmholtz-Zentrum Berlin (HZB)The furin:RVKR-CMK:Nb14 complex  using an Aekta Micro FPLC system . The proteins were premixed at an equimolar ratio or mixed with GPC buffer for control runs at final concentrations of ~23μM each and subsequently subjected to GPC at 0.2 ml/min. The peak fractions were analysed by SDS-PAGE. GPC binding assays were repeated three times.Analytical gel permeation chromatography runs were performed on a Superdex 200 5/150 GL column  in GPC buffer  with accession codes 5JMO and 5JMR, respectively.How to cite this article: Dahms, S. O. et al. The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers. Sci. Rep.6, 34303; doi: 10.1038/srep34303 (2016).


Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold β-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif. The ubiquitous small molecule, cyclic AMP (cAMP), was the first intracellular second messenger to be described and is known to play a pivotal role in regulating many key cellular processes –5. Such PDE4A/PDE4B/PDE4C/PDE4D), which generate over 20 distinct isoforms through alternate mRNA splicing and the use of distinct promoters [Members of the phosphodiesterase-4 (PDE4) cAMP-specific PDE family play pivotal roles in forming and shaping cAMP gradients within cells. These enzymes are encoded by four genes  that is unique to PDE4 long isoforms. We also uncover an additional docking site, which is located within the PDE4 catalytic unit, which is employed by an array of proteins capable of interacting with PDE4. We propose that this forms a multifunctional docking site (MFD) that, as well as aiding fidelity of interaction, confers exclusivity of binding between PDE4 and specific partner proteins.Peptide libraries were generated by automatic SPOT synthesis on Whatman 50 cellulose membranes using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry with the Autospot-Robot ASS 222 . The interaction of peptide spots with GST and GST-fused purified proteins by overlaying the cellulose membranes with 10 mg/ml recombinant protein was determined as described previously in detail ,34,35. BCOS-1 and HEK (human embryonic kidney)293 cells were propagated as previously described ,36. CellPolyFect® (QIAGEN®) was used for transfection of COS-1 and HEK293 cell lines as previously described [Immunoprecipitations were carried out as previously described in refs ,34 usingProtein samples were separated on NuPAGE 4–12% Bis–Tris polyacrylamide gels  as previously described .The proteins separated by SDS–PAGE were transferred to a nitrocellulose membrane for western blotting using the procedure outlined previously . The comWe have previously shown  that actFor the MAP kinase, Erk to phosphorylate authentic cellular substrates, such as PDE4, it binds directly to them via docking and specificity sequences that lie on each side of the phosphorylation site . With thWe then subjected lysates of Hek293 cells to immunoprecipitation using MK2-specific antisera and probed these, together with the lysates, with antisera specific for each of the PDE4B, PDE4C and PDE4D subfamilies . This alWe, and others, have successfully used a peptide array approach to delineate various protein–protein interaction surfaces  involving a specific anchor protein and a specific PDE4 isoform will thus depend on the fidelity of partnerships. However, various signalling scaffold proteins are capable of interacting with a wide range of all PDE4 isoforms. In such instances, if a particular PDE4 isoform was able to interact with more than one scaffold at a time, then this would probably lead to multimerisation into aggregates with loss of integrity of spatial compartmentalisation of cAMP degradation. Similarly, the ability of PDE4 enzymes to be phosphorylated by various kinases will depend on the availability of free docking sites. Here, through our analyses of the interaction of the functionally important long PDE4A5 isoform, we have identified one means through which multimerisation through interaction with multiple scaffolds is prevented and availability for modification by a specific regulatory kinase is effected, namely through the identification of a cohort of interaction proteins that require docking to a common site, the MFD site.The sequestration of PDE4 isoforms by signalling scaffolds invariably involves binding at two or more sites on the PDE4 . If partThus, interaction of PDE4 isoforms with MFD-binding partner proteins will define not only subpopulations that are spatially distinct but also that show attenuated susceptibility to phosphorylation by kinases such as MK2 and Erk, both of which modify the functional outcome of stimulatory PKA phosphorylation ,29.


Bullous pemphigoid (BP) is the most common autoimmune skin disease of blistering character. The underlying pathophysiological mechanism involves an immune attack, usually by IgG class autoantibodies, on the autoantigen BP 180/BPAg2, which is a type XVII collagen (COL17) protein acting as the adhesion molecule between the epidermis and the basement membrane of the dermis. About 40 years ago, following consistent findings of elevated total serum IgE levels in BP patients, it was hypothesized that IgE may be involved in the pathophysiology of BP. Our objective was to determine whether there is strong evidence for an association between IgE class autoantibodies and the clinical severity or phenotype of BP. Three databases were searched for relevant studies and appropriate exclusion and inclusion criteria were applied. Data was extracted and assessed in relation to the study questions concerning the clinical significance of IgE autoantibodies in BP. Nine studies found that anti-BP180 autoantibodies of IgE class are associated with increased severity of BP, whereas two studies did not find such an association. The number of studies which found an association between higher IgE autoantibody levels and the erythematous urticarial phenotype of BP (5) was equal in number to the studies which found no such association (5). In conclusion, higher serum IgE autoantibody levels are associated with more severe clinical manifestations of BP. There is insufficient evidence to support higher IgE autoantibody levels being associated with specific clinical phenotypes of BP. IgE has traditionally been linked to allergic Type 1 hypersensitivity reactions. However, just over four decades ago, antinuclear autoantibodies of IgE class were detected in RA and SLE patients and it was hypothesized that IgE may have a pathophysiological role in autoimmunity . This asA pathophysiological role of IgE is observed in several autoimmune diseases ranging from systemic ones, like SLE , to tissPemphigoid is a group of autoimmune disorders in which autoantibodies attack the structural proteins comprising the junction of the epidermal and dermal layers of the skin . Pemphig2 agonist treatment [Studies on BP patients show elevated IgE class autoantibodies with a pathophysiological role. Evidence lies within the detection of IgE autoantibodies targeting the BP180 adhesion molecule and leading to the manifested symptoms [reatment , has proreatment , 21.With this information in hand, we decided to carry out a systematic literature review to determine, through analysis of the studies available, whether there is validity in the findings of IgE autoantibodies in BP, and if so, whether the presence of elevated IgE autoantibody levels affects the severity of the phenotype and/or the course of the disease.MEDLINE was accessed through PubMed, EMBASE through OVID and WEB OF SCIENCE through the Wiley Online Library. Each database was searched with the term ‘(ige or immunoglobulin e) and (bullous pemphigoid or bp)’ on 15 October 2016. This yielded a total of 1084 records as follows: MEDLINE, 496; EMBASE, 31; WEB OF SCIENCE, 557. At this initial stage, for comparison between disease, searches were also performed for IgE in chronic idiopathic urticaria (656 records) and IgE autoantibodies in atopic dermatitis (281 records), giving a total of 2021 records. Figure The first findings that led to the concept that IgE may be linked to BP were published in 1974. Following this, in the 1970s and 1980s, a variety of studies were conducted to address this hypothesis. During these two decades, blood sampling and testing, immunofluorescence on lesioned skin biopsies and chromatography using dermal infiltrate were the main laboratory techniques employed in this research. As is clearly illustrated below, the role of IgE in BP during this time was only demonstrated in a non-specific fashion: for example, through findings of IgE deposition in skin basement membrane (BM), elevated serum IgE levels and peripheral blood eosinophilia. Inevitably, it was unclear whether the apparent involvement of IgE in BP was of a pathogenic nature, or whether it was simply an epiphenomenon of the disease itself.In 1974, elevated IgE levels were reported in 70% of patients with bullous pemphigoid . In the In the early 1990s, the application of newer techniques, such as ELISA, enabled better definition of the relationship between IgE and BP, as it was then possible to identify specific receptors for IgE that were possibly involved in the pathophysiology. By the mid-1990s, it was established that BP involved impaired B cell function, and the soluble form of CD23 (sCD23), a low affinity IgE receptor (FcεRII) on haematopoietic cells, was known to be involved in B lymphocyte growth and differentiation. As a result, the role of CD23 in the pathophysiology of BP was investigated and elevated levels of sCD23 were detected in the sera of BP patients . FurtherSome pivotal studies in the development of the field showed specific autoantigens in the hemidesmosomal proteins of the skin to be the targets of autoreactive IgE antibodies. As discussed below, BP180 and BP230 are major autoantigens targeted by IgE autoantibodies in BP. BP180 (BPAg2), a transmembrane protein, was the first autoantigen characterized in BP and was found to have collagenous repeats in its ectodomain capable of forming collagen-like helices ; indeed,A study in 1996 using a radioimmunoassay reported 12 out of 19 BP patients to have IgE autoantibodies against the BP55 antigen . Two yeaAn interesting recent observation concerning the pathophysiology of IgE in BP indicates that IgE may have FcR-independent effects: IgE was found to stimulate FcR-independent release of IL-6 and IL-8 by keratinocytes in vitro . When thA highly sensitive method for detecting anti-BP180 NC16A IgE autoantibodies in sera of BP patients was reported in 2009 . The assMore recently, Pomponi et al. evaluated the efficiency of the ISAC® microarray system in detecting IgE and IgG antibodies in BP patients . The exp3 . Four months after discontinuation of treatment, the patient reported pruritus and new bullae formation, but re-initiation of omalizumb resulted in resolution of bullae and subsidence of pruritus. This was the first treatment targeting IgE in a BP patient and it was also the first in vivo demonstration of the pathogenicity of IgE class autoantibodies in BP. Similarly, in 2012, Dufour et al. [The hypothesis of IgE having a pathogenic role in BP has been further established through the successful treatment of BP patients with omalizumab, a humanized monoclonal anti-IgE antibody which binds to IgE . In 2009r et al. . Five ofAs the role of IgE in the pathophysiology of BP became better understood as a pathogenic one, hypotheses about how its presence in BP may affect the disease severity/activity and/or clinical manifestations (i.e. phenotype) were put forward. Of the earliest papers to explore a possible correlation between IgE and BP activity was that of Asbrink and Hovmark , which sDisease severity of BP has been defined in a variety of ways over the years. The severity of BP used to be assessed by counting the number of bullae on the skin of the patient. Joly et al. and Bernard et al. , 33 repoAs shown in Table Since it has been demonstrated that total serum IgE levels correlate directly with the levels of IgE specific for BP180 and BP230 antigens  and thatOverall, the high proportion of studies reporting an association between anti-BP180 IgE autoantibodies and severity of BP compared to the studies reporting no such association (9:2) favours a correlation between the presence of IgE autoantibodies and increased disease severity.Bullous pemphigoid does not manifest as a single phenotype. The typical bullous phenotype involves local or widespread blisters and eruptions; however, these can be preceded by lesions of an urticarial nature on erythematous skin. One of the less common forms of BP is pemphigoid nodularis (nodular phenotype) which is characterized by highly pruritic lesions, often formed on nodular skin .Table Amber  suggesteFirstly, whereas IgG-mediated murine models lack urticarial erythema and eosinophil infiltration, these features occur in human skin engrafted on to SCID mice inoculated with monoclonal IgE specific to the BP180 ectodomain . A similSecondly, autoantibodies of IgE class have been reported in patients with other inflammatory skin diseases. IgE autoantibodies specific for double-stranded DNA  and for As shown in Table This review’s results support the conclusion that the higher the serum IgE autoantibody levels are, the more severe the manifestation of BP will be; however, they do not support the possibility that higher IgE autoantibody levels promote a more urticarial presentation of BP. It is fair to say that there is ambiguity as to whether IgE can be held accountable for the full spectrum of BP manifestations, or whether its effects are more relevant to the initial stages of the disease. Further research is needed in the future to establish more solid conclusions. There is a need for more multi-centre prospective cohort studies which would help to eliminate uncertainties in the field, particularly with respect to a possible association between the occurrence of IgE class autoantibodies and the clinical phenotype of BP.


GSK3β is thought to be constitutively activated by autophosphorylation at Tyr216 and inactivated by phosphorylation at Ser9. The GSK3β activity has previously been evaluated by inhibitory Ser9 phosphorylation, but it does not necessarily indicate the kinase activity itself. Here, we applied the Phos-tag SDS-PAGE technique to the analysis of GSK3β phosphoisotypes in cells and brains. There were three phosphoisotypes of GSK3β; double phosphorylation at Ser9 and Tyr216, single phosphorylation at Tyr216 and the nonphosphorylated isotype. Active GSK3β with phosphorylation at Tyr216 represented half or more of the total GSK3β in cultured cells. Although levels of phospho-Ser9 were increased by insulin treatment, Ser9 phosphorylation occurred only in a minor fraction of GSK3β. In mouse brains, GSK3β was principally in the active form with little Ser9 phosphorylation, and the phosphoisotypes of GSK3β changed depending on the regions of the brain, age, sex and disease conditions. These results indicate that the Phos-tag SDS-PAGE method provides a simple and appropriate measurement of active GSK3β  GSK3β is ubiquitously expressed in all types of cells and tissues and is particularly abundant in the brain. GSK3β regulates many cellular functions, including cell proliferation, cell survival, gene expression, cellular architecture, neural development and plasticity etc.4. In addition to these physiological functions, GSK3β is also involved in cancers, diabetes and neurodegenerative diseases. In Alzheimer’s disease (AD), GSK3β produces pathological and abnormal phosphorylation of tau7, but it is not known how GSK3β becomes to phosphorylate tau abnormally in AD brains. It is important to understand the mechanisms that regulate the kinase activity of GSK3β to clarify its versatile functions.Glycogen synthase kinase 3β (GSK3β) is a multifunctional protein kinase that targets Ser/Thr residues with a priming phosphorylation at the fourth amino acid on the C-terminal sidein vitro4. GSK3β requires phosphorylation at Tyr216 in the activation loop for activation9. Although GSK3β is a Ser/Thr kinase, this Tyr phosphorylation is thought to be autophosphorylation10; however, phosphorylation by Src family tyrosine kinases has also been reported11. Tyr216-phosphorylated GSK3β is constitutively active and is inactivated by phosphorylation at the N-terminal Ser9 by several protein kinases, such as PKA, PKB, p90RSK and Akt13. Phosphorylated Ser9 binds to a pocket for a priming phosphorylation in the substrate-binding region and to reduce the binding affinity for substrates15.The regulation of GSK3β activity is well studied de novo synthesis or if they require any signal for activation. On the other hand, many signaling pathways are reported to downregulate GSK3β activity by phosphorylation at Ser913. For instance, insulin and other growth factors activate Akt, which in turn phosphorylates GSK3β at Ser9, rendering the kinase inactive and resulting in decreased phosphorylation of downstream substrates, such as glycogen synthase, tau, β-catenin, etc.16. Although some reports measured reduced GSK3β activity in cultured cells upon stimulation with growth factors17, no simple method is available to estimate the amount of active GSK3β in vivo.However, the cellular mechanisms that regulate GSK3β are not yet fully understood. For example, it is not known what amount of GSK3β remains inactive without Tyr216 phosphorylation. Therefore, it is unclear whether all GSK3β molecules are automatically activated by autophosphorylation after 13. However, it is hard to correctly estimate the kinase activity using the levels of inhibitory phosphorylation. The immunoblotting data showing an increase in the phospho-Ser9 levels in cells after growth factor treatment do not provide the absolute extent of inactivation. For example, a 2-fold increase in the phospho-Ser9 levels did not discriminate the 1 to 2% or 50 to 100% changes in the total GSK3β levels. We thought that it would be much more informative to measure the active form of GSK3β if possible. We have previously used Phos-tag SDS-PAGE, which was first reported by Kinoshita et al.18, to reveal the in vivo phosphorylation states of p35 Cdk5 activator, and indicated the usefulness of the method in quantitatively measuring the phosphoisotypes of proteins19. In this study, we measured the absolute amount of active GSK3β in cultured cells, primary neurons and mouse brains using the Phos-tag technique. In fact, we could measure the active form of GSK3β in vivo and found that the amount of active GSK3β changed in brains depending on the regions, ages, sex and disease conditions.Because it was shown that phosphorylation at Ser9 inhibits GSK3β activity, most previous studies have used the increase or decrease in the phospho-Ser9 levels as changes of cellular GSK3β activityWe expressed GSK3β in CHO-K1 cells and examined its separation on Phos-tag SDS-PAGE to determine the phosphoisotypes of GSK3β. Although GSK3β appeared as a single band at 47 kDa on Laemmli’s SDS-PAGE gels Fig. . These mWe immunoblotted the three GSK3β bands on Phos-tag SDS-PAGE with anti-phospho-Ser9 (pS9) or anti-phospho-Tyr216 (pY216) antibodies to confirm the results described above. The anti-phospho-Ser9 antibody only reacted with the upper band and the anti-phospho-Tyr216 antibody recognized both the upper and middle bands  in culture  and insulin on GSK3β activity in primary neurons at 5 DIV31.The degree of phospho-Ser9 was decreased with maturation of cultured neurons Fig. . Then weWe then analyzed the phosphorylation of GSK3β in brains of aged male and female mouse at age 1.5 years, using four different regions; cerebral cortex, cerebellum, olfactory bulb and hippocampus Fig. . After c33. Then, we analyzed the GSK3β phosphoisotypes in the brains of db/db mice, a model of Type 2 diabetes33, using the Phos-tag method. The db/db mouse displays hyperglycemia and insulin-resistance. There are three reports on GSK3 activity in the db/db mouse brain with different results: increased Ser9 phosphorylation33 and no change in phosphorylation34. It is recently reported that Ser9 phosphorylation increased at 4 weeks, but not 26 weeks in db/db mice35. The phosphoisotypes were examined in the brains of 11-week-old male mice using Laemmli’s  and cerebellum of aged female mouse. These results could also be observed clearly and firstly by the use of the Phos-tag method. The differences of GSK3β phosphoisotypes between sexes or among brain regions may be related to age-dependent changes in hormone levels, such as estradiol or insulin-like growth factor44.It is accepted that cellular GSK3β is active upon phosphorylation at Tyr216 in the activation loop42. We treated cells with insulin to determine the extent of the increased Ser9 phosphorylation of GSK3β. Consistent with previous many reports, Ser9 phosphorylation increased approximately 0.5-fold upon insulin treatment when it was examined by immunoblotting with the anti-phospho-Ser9 antibody. A similar extent of increased phosphorylation was obtained, even when the upper Ser9-phospho-band in Phos-tag was measured, indicating that the Phos-tag method measures the relative changes of Ser9 phosphorylation properly. However, the increase was rather small when the proportion of phospho-Ser9 GSK3β was evaluated against the total GSK3β molecule. For example, in PC-12 cells, the population of GSK3β with Ser9 phosphorylation was increased almost 2-fold by insulin treatment , Phe mutant at Tyr216 (Y216F), and double mutant at Ser9/Tyr216 (AF) were generated by site-directed mutagenesis using mouse GSK3β as a template. The primers used in this study were: 5′-CGGCCCAGAACCACCGCCTTGCGGAGAGC-3′ and 5′-GCTCTCCGCAAAGGCGGTGGTTCTGGGCCG–3′ for S9A, and 5′-GAACCCAATGTTTCGTTTATCTGTTCTCGG-3′ and 5′-CCGAGAACAATAAACGAAACATTGGGTTC-3′ for Y216F. The mutations were confirmed by DNA sequencing.2, 10 mM NaF, 10 mM β-glycerophosphate, 10 μg/ml Pefabloc, 10 μg/ml leupeptin and 1 mM dithiothreitol. After centrifugation at 18,000 × g for 20 min, the supernatant was used as the brain extract. db/db mice, a model of Type 2 diabetes, and age matched non-diabetic wild type (WT) mice were obtained from CREA Japan, Inc. . Cerebral cortices were dissected from an 11-week-old male mouse and the brain extract was prepared as described above for the ICR mice.All animal experiments were performed according to the guidelines for animal experimentation of Tokyo Metropolitan University (in accordance with the Society for Neuroscience Guidelines). The study was approved by the Research Ethics Committee of Tokyo Metropolitan University . All efforts were made to reduce the sufferings of animals used. ICR mice at different age groups were obtained from Sankyo Laboratory Service . The mouse was housed in a temperature-controlled room under 12 h light/dark cycle with free access to food and water. Mouse brains were dissected into cerebral cortices, cerebella, olfactory bulbs and hippocampi immediately after decapitation. The brain regions were homogenized using a Teflon-pestle homogenizer in 10 vol (v/w) of 20 mM HEPES, pH 7.4, 1 mM EGTA, 100 mM NaCl, 0.1% Nonidet P-40, 2 mM MgCl5 cells/ml and treated with SB415286  at 20 μg/ml or LiCl at 10 mM for 5 and 10 h. For insulin study, the culture medium was replaced with serum-free media for 6 h, and then insulin  was added at 50 µU/ml to the medium for 10, 30 and 60 min.CHO-K1, HEK293, COS-7, SH-SY5Y and PC-12 cells were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml Streptomycin. CHO-K1 cells were transfected with a plasmid encoding GSK3β WT or the S9A, Y216F or AF mutant using Lipofectamine 2000 , or HilyMax  according to the manufacturer’s instruction. All transfections were performed in triplicate. To study the effect of GSK3β inhibitors, CHO-K1 cells were plated at a density of 1 × 106 cells/ml50. The medium was changed to Neurobasal medium supplemented with 2% B-27 , 0.5 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin after 4 h of plating. Half of the medium was replaced with fresh medium every third day. For Insulin-like growth factor (IGF) or insulin treatment, primary neurons at 5 DIV were cultured in the absence of B-27 for 12 h, and then IGF-1  and insulin were added at 100 ng/ml and 50 μU/ml respectively, in the medium for 10, 30 and 60 min, followed by cell collection.Primary neurons were prepared from the mouse brain cortex at embryonic day 16 (E16) and plated on polyethyleneimine-coated dishes in DMEM and Ham’s F-12 (1:1) supplemented with 5% fetal bovine serum, 5% horse serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin at a density of 1.5 × 102, 10 μg/ml leupeptin, 0.4 mM Pefabloc and 0.5% (v/v) Nonidet P-40. The lysate was centrifuged at 18,000 x g for 15 min, the supernatant was collected and incubated with 200 μg/ml anti-HA (Y-11)  for transfected GSK3β-HA or its mutants and 100 μg/ml anti-GSK3β  for endogenous GSK3β along with 5 μl {50% (v/v) slurry} of Dynabeads Protein G  by rotation for the respective experiments. Beads were washed in the same buffer. The kinase activity of the immunoprecipitates was measured using a GSK3β peptide  and [γ-32P]ATP as substrates in kinase buffer  for 5 min by Cerenkov counting in a liquid scintillation counter . The amino acid sequence of the GSK3β peptide is YRRAAVPPAPSLSRHSSPHQSpEDEE (singer letter representation), in which the prime phosphorylation site is Ser at fifth amino acid from the C-terminus51.CHO-K1 cells expressing GSK3β-HA or its mutants and HEK293 cells treated with insulin were lysed in 20 mM Hepes, pH 7.4, 0.5 mM EGTA, 0.1 mM EDTA, 0.15 M NaCl, 1 mM MgCl2, 100 mM NaCl, 0.5% Nonidet P-40, 0.4 mM Pefabloc, 10 μg/ml leupeptin and 1 mM dithiothreitol on ice for 5 min. After centrifugation at 18,000 × g for 15 min, the supernatant was collected as the cell extract. The extract was incubated with 2.5 U/ml Escherichia coli. alkaline phosphatase  in 50 mM Tris, pH 8.8, 0.15 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA and 1 mM MgCl2 at 37 °C for 3 h.CHO-K1 cells expressing GSK3β were lysed in 50 mM Tris HCl, pH 8.0, 1 mM MgCl2, 100 mM NaCl, 0.5% Nonidet P-40, 0.4 mM Pefabloc, 10 μg/ml leupeptin, 10 mM Sodium Fluoride, 10 mM β-Glycerophosphate and 1 mM dithiothreitol on ice for 5 min52. After centrifugation at 18,000 x g for 15 min, the supernatant was collected and the protein concentration was measured using Coomassie Protein Assay reagent  followed by denaturation. Laemmli’s SDS-PAGE was performed using 10% (w/v) polyacrylamide gels. Proteins were transferred to PVDF membranes  using a semidry blotting apparatus. The membranes were probed with antibodies against GSK3β , p-S9 GSK3β , p-Y216 GSK3β , Actin , Akt , p-Akt  , p-β-Catenin , GSK3α . The appropriate horseradish peroxidase-conjugated secondary antibodies  were then applied. The immunoreactions were developed with the Immobilon Western Chemiluminescent HRP substrate  and the immunoreactions were captured into a FUSION SL apparatus  using an automated ‘paste marker’ function.The cells were washed with 1x PBS and lysed in 20 mM HEPES, pH 7.4, 1 mM MgClet al.18. Phos-tag acrylamide was purchased from Wako Chemicals . Separating gels were made by copolymerization of acrylamide with Phos-tag acrylamide. The migration of phosphoproteins in the separating gel are less than those of their nonphosphorylated counterpart because the Phos-tag traps phosphoproteins reversibly during electrophoresis (see Kinoshita et al., Nat Protocol., 2009 for a ref. 254 in 10 mA current. This makes the separation of proteins possible by detecting shifts in the mobility of phosphoproteins and identified as various bands based on the degree of phosphorylation. The separated proteins were transferred to PVDF membranes using a submerged blotting apparatus. Immunoreaction of the membrane is carried out as described above.Phosphorylation states of GSK3β were examined using Phos-tag SDS-PAGE, which is a phospho-affinity SDS-PAGE developed by Kinoshita t-test or one–way ANOVA using GraphPad PRISM software version 5.0. P < 0.05 was considered significantly different.The band intensities and banding profiles were analysed with Image J software. The data are represented as mean ± s.e.m. from more than three independent set of experiments. Statistical significance was determined by Student’s Supplementary Information


The present study aims to investigate inhibition in individuals with Down Syndrome compared to typically developing children with different inhibitory tasks tapping response inhibition and interference suppression. Previous studies that aimed to investigate inhibition in individuals with Down Syndrome reported contradictory results that are difficult to compare given the different types of inhibitory tasks used and the lack of reference to a theoretical model of inhibition that was tested in children (see Bunge et al.,  Down Syndrome (DS) is the most common genetic syndrome associated with intellectual disability and affects ~1 in 700 newborns , were included in the DS group. Thirty-five typically developing children, 18 girls and 17 boys with a mean age of 5 years and 6 months , were included in the typically developing control group of 5-year-olds (5TD). Thirty typically developing children, 13 girls and 17 boys with a mean age of 6 years and 2 months , were included in the typically developing control group of 6-year-olds (6TD). Individuals with DS had trisomy 21 without mosaicism and were recruited from two treatment centers in the north of Italy. Typically developing children were recruited from different educational services in the same area. None of the children had a history of neurological impairment or developmental disabilities.A final sample of 97 individuals belonging to three groups took part in this study. Thirty-two individuals with Down Syndrome (DS), 22 girls and 10 boys with a mean age of 14 years and 4 months  was 0.55, p < 0.0005  were 0.67 for PMFFT Errors and 0.95 for PMFFT Time  and the mean latency between the presentation of the item and the child's response  were recorded. Cronbach's alphas calculated in a sample of 174 children  was 0.42, p = 0.002 and 0.56, p < 0.001 for Flanker Accuracy and Flanker Time, respectively  was 0.62 (p < 0.001) for Dots Accuracy and 0.72 (p > 0. 001) for Dots Time , the root mean square error of approximation (RMSEA), the standardized root mean squared residual (SRMR), the Akaike Information Criterion (AIC), and the Bayesian information criterion (BIC). The X2 test was used to evaluate the appropriateness of the CFA model. Non-significant X2 values indicated a minor difference between the covariance matrix generated by the model and the observed matrix and thus an acceptable fit. CFI values > 0.97 are indicative of a good fit, whereas values > 0.95 may be interpreted as an acceptable fit  was performed using the TD group's inhibitory task scores to verify the characteristics of the inhibition construct in early childhood. Multiple fit indices were considered to compare models , Flanker Accuracy and Dots Accuracy. The DS group showed high variability in all tasks. This group performed worse than the 6TD group but was similar to the 5TD group in PMFFT accuracy. The opposite was observed for PMFFT time, and the DS group showed a similar response time to the 6TD and a higher response time than 5TD. A significant difference emerged in the Go/No-Go task between the 6TD and DS groups; however, this difference disappeared when a mathematical transformation  and the 6-year-old (r ranged from 0.391 to 0.754) groups. However, in the DS group, only the Dots Accuracy and the Dots Time scores were related (r = 0.372). The CPM performance was associated with the PMFFT Time and the Flanker task (Time and Accuracy) in the 6TD group, no significant association emerged considering the 5TD group, and CPM was related to the PMFFT Time in the DS group. Finally, age was significantly related only to the PMFFT time in the 5TD group.As expected, the inhibition task scores were not highly related .The unitary model had mediocre or unacceptable fit indices: χF = 8.363 p < 0.001, and interference suppression, F = 10.530 p < 0.001. The 6TD group outperformed the 5TD group in both the response inhibition  and interference suppression  components. No differences were found in either inhibition component between the SD and 5TD groups. In contrast, the 6TD group outperformed the DS group on the response inhibition component score  and the interference suppression component score .Two composite scores representing response inhibition and interference suppression were calculated as the mean of the z-scores as follows: the z-score average of PMFFT Errors and Go/No-Go task Accuracy for response inhibition and the z-score average of Flanker Accuracy and Dots Accuracy for interference suppression Table . These cThe main goal of this study was to investigate diverse inhibition components in children and youth with DS compared to two groups of typically developing children aged 5 and 6 years matched for mental age. Specifically, we aimed to focus on response inhibition and on interference suppression components  and in the Flanker Task accuracy. Moreover, the DS group had a higher response time than the 5-year-olds on the Preschool Matching Familiar Figure Task and a higher response time than both control groups on the Flanker task. This inconsistent pattern is in line with the inhibition literature . Previous studies considered both accuracy and response time, and, as in our study, mixed results were reported. Nevertheless, it must be noted that in our study, accuracy was unrelated to response time in both the Flanker task and the Preschool Matching Familiar Task. This evidence may suggest that as early pre-schoolers (Traverso et al., We speculate that focusing on single task differences makes it difficult to investigate the efficacy of the inhibition components (see Miyake et al., In summary, our findings demonstrate that individuals with DS show a delay in inhibition development, but their performance is similar to the typical development of 5-year-old children. This evidence is consistent with the study by Borella et al. , in whicTo the best of our knowledge, in the last 20 years, only ten studies have examined the inhibition abilities of individuals with DS. These studies reported contradictory results and generally used only response inhibition tasks without referring to a theoretical model of inhibition (see Borella et al., There were some weaknesses in the current study that should be noted. First, although this study aimed to focus on inhibition, it would have been useful to control for other non-executive or executive abilities, such as working memory. Second, although the formative indices may represent a useful methodology to investigate executive functions, in this study, after testing the inhibition model on typical-developmental children with an EFA, we assumed that the inhibition construct was similar in both typical and atypical development. Increasing the sample size would be useful to examine findings observed using reflective and formative inhibition indices (Willoughby et al., This study was carried out in accordance with the recommendations of the Ethical Code of Italian Psychology Order and of the Ethical guidelines of the Italian Association of Psychology with written informed consent from all subjects. All parents of the subjects gave written informed consent in accordance with the Declaration of Helsinki. At the time we collected the data no ethical committee was yet present to which we could refer to.LT and MU: revised the literature on inhibition development; MF and MP: revised the literature concerning inhibition in individuals with Down Syndrome; MU, MP, LT, and MF: conceived and designed the experiment; LT and MF: collected the data; LT and MU: performed the analysis; LT: wrote a first draft of the manuscript that was revised by MF, MU, and MP. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB β-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B β-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-β7 and β11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 β-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens.We analyzed the kinetic properties of the metagenomic class B3 β-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-β-lactamases (MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on  This global commitment to develop action plans on antimicrobial resistance was reaffirmed this year at UN based on the “Global Action Plan on Antimicrobial Resistance” .Antimicrobial resistance is a worrisome issue in healthcare worldwide during the last two decades. This led the WHO to declare April 7Among the frightening arsenal of resistance mechanisms we face today, β-lactamases still represent the most relevant threat. The increasing prevalence of enzymes like the carbapenemases NDM-1 and KPC , and theMetallo(zinc)-β-lactamases or class B β-lactamases (EC 3.5.2.6) belong to a protein superfamily including more than 6,000 members that share a common fold and a handful of conserved motifs. They are divided into at least 15 families primarily based on their biological functions, for which the classification does not strictly reflect their phylogenetic relationship , 10. ZinSerratia and Aeromonas  are the reporter substrate’s Km and fixed concentration used, respectively.For low er et al . For com482 = +15,000 M-1.cm-1), benzyl-penicillin (Δε235 = –775 M-1.cm-1), ampicillin (Δε235 = –820 M-1.cm-1), piperacillin (Δε235 = –820 M-1.cm-1), cephalothin , cefuroxime , cefoxitin , ceftazidime , cefotaxime , cefepime , imipenem , meropenem , ertapenem (Δε300 = –775 M-1.cm-1) and aztreonam (Δε318 = –750 M-1.cm-1).The following extinction coefficients and wavelengths were used: nitrocefin (Δε50 was assessed as the chelating agent’s concentration able to inhibit 50% LRA-12 activity.Inhibition of enzymatic activity by ethylenediaminetetraacetic acid (EDTA) and pyridine-2,6-dicarboxylic acid  was assayed by measuring the hydrolysis of 170 μM ertapenem after incubating the enzyme for 20 min at 25°C in the presence of different concentrations of EDTA, following residual activity of LRA-12 in 50 mM sodium phosphate buffer (pH 7.5); for DPA, hydrolysis of ertapenem was measured immediately after the addition of the different concentrations of the chelating agent. The inhibition parameter ICCrystals were grown at 20°C by hanging drop vapor diffusion with drops containing 0.2 μL of LRA-12 solution (15 mg/ml) and 0.2 μL of 0.2 M ammonium sulfate, 0.1 M Tris pH 8.5, 25% w/v polyethylene glycol (PEG) 3350.Data were collected at 100 K on a Dectris (Pilatus 6M) detector at a wavelength of 0.97857 Å on Proxima 1 beamline at the Soleil Synchrotron . Indexing and integration were carried out using XDS , and theStructure modeling was achieved by molecular replacement through Phaser and Buccaneer pipeline in CCP4 suite , using Fwww.pymol.org) [Model visualization and representation was performed with WinCoot 0.8.2 , and PyMmol.org) . Topologmol.org) . Predictmol.org) .The coordinates and structure factors of the native LRA-12 β-lactamase were deposited under accession code 5AEB (released: 2015-09-16).Elizabethkingia meningoseptica (formerly known as Chryseobacterium meningosepticum) [The predicted amino acid sequence of the metagenomic metallo-β-lactamase (MBL) LRA-12 from Alaskan soil revealed a high percentage amino acid identity with previously described MBLs from environmental origin. It presented 60–69% amino acid identity with GOB MBLs from epticum) , includiepticum) , with whFluoribacter gormanii) and BJP-1 (Bradyrhizobium japonicum), respectively, two class B3 enzymes for which the crystallographic structures have been solved [The MBL also showed 41% and 33% identity with FEZ-1 . For carbapenems, the clone was also resistant to meropenem , but susceptible to imipenem, although the MIC value for this drug (1 μg/mL) was about 16/32-fold higher than the MIC for recipient E. coli control strains .Compared to the reference 0 strain , the cloi clones . The E.  strains  and, as  present . As expekcat/Km values ranging from 0.16 to 1.75 μM-1.s-1. The enzyme exhibited higher apparent affinity for cephalothin, cefuroxime and cefoxitin . For cephalothin and cefoxitin, accompanying low turnover rates  result in the highest catalytic efficiency values, equivalent to that of LRA-12 acting upon imipenem. High kcat/Km values were also observed for LRA-12 acting upon all three carbapenems evaluated, imipenem, meropenem and ertapenem , in contrast to its behavior with oxyimino-cephalosporins, with which it displayed lower catalytic efficiencies . As expected for metallo-β-lactamases [Ki of 146 μM.The kinetic parameters of LRA-12 were determined for a representative group of β-lactams . ResultsKinetic parameters were compared to those reported for other class B3 metallo-β-lactamases . MetagenActivity of LRA-12 in presence of two different chelating agents was assessed by two approaches.Pre-incubation of LRA-12 with 50 mM EDTA inactivate 91% of the enzyme within 20 min  and EDTA50 of 113 μM. The IC50 for DPA was 460 μM. These results suggest that DPA was a stronger inhibitor than EDTA, as DPA did not require pre-incubation; however, the IC50 value for EDTA was lower than that for DPA.EDTA displayed an IC1 1, and crystals diffracted at a final resolution of 2.1 Å. Main data and refinement statistics are shown in Metagenomic LRA-12 crystallized in space group P 1 2The refined structure consists in two monomers per asymmetric unit. Monomer A includes 264 amino acids of the 269 residue long mature β-lactamase, from Val28 to Asn306; monomer B is composed of 266 residues, from Gln26 to Asn306, following BBL numbering . Both moThe overall structure of LRA-12 conserves the main structural features of a class B β-lactamase , showingThe root mean-square deviation (rmsd) between the equivalent Cα atoms in both monomers is 0.31 Å and no significant difference is found between the two active sites. Due to this observation, further discussion will refer to both monomers unless otherwise noted.LRA-12 structure is very similar to other class B3 β-lactamases, despite low sequence identities within the sub-class , 49, 56.A 23-residue-long loop (Asp148-Arg172) with apparent high mobility, between α3 and β7, is present at the entrance of the active site. This loop is also found in other B3 β-lactamases, although some of them like GOB-18, FEZ-1 and BJP-1 include a short α-helix within this loop . There iAn important difference between LRA-12 and other B3 enzymes like FEZ-1, L1 and BJP-1 is the absence of disulfide bridges in LRA-12. In FEZ-1 and L1, a disulfide bridge occurs between Cys256 and Cys290 (FEZ-1) or Cys296 (L1); these cysteines are replaced by aromatic residues Phe256 and Tyr290, respectively. Also, Cys181 and Cys201, creating the disulfide bridge in BJP-1, are replaced by Ser200 and Asn220, respectively, in LRA-12. Metagenomic β-lactamase LRA-12 has only two cysteine residues not involved in disulfide bridges: Cys70 ; and Cys201, also present in GOB-18 (Cys180), which is buried within the β-lactamase core and associated through hydrogen bonds with backbone carbonyl groups of Thr114 and Thr115, located at the β5-α3 loop.Interestingly, a cobalt ion was modeled between the two monomers of LRA-12 in the asymmetric unit, bound to Glu140-Oε and His175-Nε2 in a tetrahedral coordination. This cobalt, present in the crystallization buffers, could help in the crystallization process by stabilizing intermolecular interactions, as previously suggested .Finally, five sulfate ions (2 in monomer A and 3 in monomer B) were also modeled, all bound to solvent-exposed residues and not interfering with important residues from the active site cavity.3, similar to what was reported for GOB-18 (150–200 Å3) [3), BJP-1 (ca. 110 Å3), and L1 (ca. 70 Å3). These findings are in agreement with the aforementioned discussion about the equivalent catalytic behavior of LRA-12 and GOB β-lactamases, compared to other B3 β-lactamases [The active site of LRA-12, as in the other metallo-β-lactamases, is located between the two “αβ” moieties of the αββα fold, above the β-sheets, covered by the flexible α3-β7 loop, and contoured by the 10 amino acids N-terminal segment, the β11-α5 loop, and the α4. The catalytic cavity has a tridimensional geometry of a shallow cleft having 150–170 Å–200 Å3) . The volctamases , and couThe β-lactamase LRA-12 contains two heavy atoms in the active site of each of the two monomers in the asymmetric unit, according to well-defined electron densities. This is consistent with the fact that most of the class B3 β-lactamases contain two zinc ions in the active site, constituting what is known as the metal- or zinc-binding site. Comparative constitution of zinc-binding sites with other MBLs is shown in The metal-binding site 1 is defined by Gln116, His118, and His196 (site QHH), which is uniquely found in this enzyme and in GOB β-lactamases ; zinc-biThe zinc ion at the QHH site (Zn1308 in the PDB) is bound to Gln116-Oε1, His118-Nδ1, His196-Nε2, and two water molecules, Wat2045 and Wat2040 in the PDB coordinate file. The latter constitutes the nucleophilic water molecule that serves as a bridge between both Zn(II) ions, as observed in most of the class B3 MBLs, providing one of the binding points with zinc ion at DHH site (Zn1307 in the PDB), which is also bound to Asp120-Oδ2, His121-Nε2, His263-Nε2, and water molecule Wat2041 .Also, intra- and inter-metal site interactions between residues from both metal sites also exist: Asp120-Oδ2 with His263-Nε2 (site 2 –site 2), His118-O with Asp120-N (site 1 –site 2), His118-O with His121-N (site 1 –site 2), and His121-Nε2 with His263-Nε2 (site 2 –site 2).Both Zn(II) ions are separated by a Zn-Zn distance of 3.60 and 3.66 Å in monomer A and B, respectively, which is equivalent to distances observed in other dinuclear B3 MBLs , 56, 58.Active site residues participating in the direct coordination of Zn(II) ligands are also stabilized by hydrogen bonds with outer sphere residues. In zinc-binding site 1, Gln116-Nε2 interacts with Asn220-Nδ2 and a water molecule, His118-Nε2 with a water molecule, and His196-Nδ1 with Thr223-Oγ1. In metal-binding site 2, His263-Nδ1 interacts with Asp67-Oδ2, and His263-O with Ser265-N and Gln266-N (not shown).S. maltophilia’s L1 (and probably FEZ-1 and BJP-1), Ser221 does not directly interact with the Zn(II) but with a second water molecule in the active site which could act as proton donor during catalysis [A remarkable difference between LRA-12 and other class B β-lactamases is the presence of a methionine residue at position 221 that is not involved in the coordination of the metal-binding site. As shown in atalysis ; insteadIn LRA-12, a water molecule (Wat2042) occupies the position of Ser221 lateral chain in FEZ-1/BJP-1, forcing the side chain of Met221 to point outwards from the active site, which enables a displacement of the β11-α5 loop (containing Met221) that carries it, thus increasing the size of the cavity; compared to FEZ-1, BJP-1, and L1, the Cα of residue at 221 is displaced 2.70, 2.72 and 3.06 Å, respectively.As in GOB-18, Met221 of LRA-12 is located within a predominantly hydrophobic cavity, without any direct interaction with the catalytic residues, and its role has been postulated to be important in protein stability . MoreoveIn this study, we report the phenotypic, kinetic and structural analysis of a metallo-β-lactamase isolated by metagenomics from an uncultured bacterium from Alaskan soils.Elizabethkingia meningoseptica GOB enzymes. This subclass includes mostly chromosomally-encoded β-lactamases from environmental microorganisms, although they have also been isolated occasionally in clinical settings .LRA-12 is a class B3, di-zinc dependent metallo-β-lactamase with highest amino acid identity to the Our results support the hypothesis that many β-lactamases originate on bacterial chromosomes. There is an increasing frequency of reports that provide evidence that the resistome contained in environmental microorganisms is a source of enzymes with native wide-range activity spectrum towards most β-lactams, even before being recruited and disseminated among human pathogens.Metallo-β-lactamases are part of a vast family of proteins with a stable scaffold that were able to evolve divergently toward diverse activities, and this evolution relied on the variation in the sequence and length of specific loops and other domains that enabled adaptation of their catalytic cavities to different types of substrates, expanding their functions within the Bacteria as well as the Eukaryotes.The environmental metagenome is a rich source of MBLs presenting both biochemical and structural similarities to other widely distributed clinically-relevant carbapenemases.blaLRA-12 gene is embedded in a ~30 kb genetic platform lacking any putative recombination/transposition signal (data not shown), which, considering that the metagenomic sample comes from an area with low exposure to antibiotics introduced by people, the possibility of occurrence of gene exchanges and selection could still be low.The structure of LRA-12 suggests that MBLs occur in environments independent of local antibiotic use. The high use of antibiotics in clinical settings as well as in livestock activities likely favored the recruitment of “silent” genes contained in culturable or unculturable microorganisms in a specific environment, and are prone of being disseminated to pathogens by horizontal gene transfer. The


Identification of over 1600 phosphoproteins and analysis of phosphorylation site motifs provide strong support for extensive protein phosphorylation and the potential regulatory roles for such modifications during anther development. Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development.As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as  In flowering plants, the development of the male reproductive organ (stamen) is a complex process involving anther primordium initiation and early cell divisions, differentiation of anther tissues, generation of haploid microspores, as well as formation and elongation of filaments. A mature anther typically has four lobes; each lobe contains the central microspore mother cells (Ms) and somatic tissues, which include four separate sporophytic cell layers, the epidermis, endothecium, middle layer, and tapetum. Anther development in Arabidopsis can be divided into two phases and 14 stages based on the morphological changes of the tissue and cells visualized by light microscopy . During EXCESS MICROSPOROCYTES1 , encoding a receptor-like kinase, was the first such gene shown to play critical roles in determination of anther cell fate during early stages of anther development (SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE1 (SERK1) and SERK2, two functionally redundant genes encoding receptor-like kinases, were found to be important for tapetum formation (BARELY ANY MERISTEM1 (BAM1) and BAM2, encoding receptor-like kinases similar to CLAVATA1, are required for the specification of somatic cells during early stages of anther development (RECEPTOR-LIKE KINASE2 (RPK2) is needed for the cell fate specification of the middle layer during the differentiation of secondary parietal cells into endothecium and middle layer cells (ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 encode leucine-rich-repeat receptor-like kinases and mitogen-activated protein kinase (MAPK) 3 and MAPK6 function in similar fashion to regulate normal anther lobe formation and anther cell differentiation  and later exine pattern formation and pollen release  and transcription factors (TFs), play crucial roles in anther development during different stages . EXCESS Transcriptional regulation is the most common and fundamental regulation during development. In addition, the activities of transcriptional factors (TFs) are often affected by post-translational modification, which coordinates various cellular processes. Reversible protein phosphorylation through protein kinases and phosphatases is one of the most widespread and important post-translational modifications in eukaryotes, often affecting the transcriptional regulatory network through phosphorylating or dephosphorylating TFs. For instance, in Arabidopsis phosphorylation of a WRKY TF (WRKY33) by MAPK3/MAPK6 is required for pollen development and function  and another close to maturity (stages 8–12). The proteomic and phosphoproteomic results reported here provide a global survey on protein expression and phosphorylation profile during Arabidopsis anther development. The results can facilitate the understanding of physiological functions underlying Ser/Thr/Tyr phosphorylation and signaling networks in anthers and related biological process.Arabidopsis thaliana [ecotype Columbia (Col)] was grown in Metro-Mix 200 soil under long-day conditions (16h light–8h dark) in a growth chamber at a constant 22 °C. We selected 3- to 4-week-old plants to collect anthers at stages 4–7 and 8–12 as previously described . The suspension was sonicated for 15min  and the supernatant was collected by centrifugation at 20 000×g for 20min. The proteins reduction, alkylation and digestion were performed as described previously  and 0.1% formic acid in 90% acetonitrile (solvent B). The peptide separation was accomplished by three-step elution: 2–35% solvent B for 200min, 35–90% solvent B for 10min, 90% solvent B for 5min, 90−2% solvent B for 2min, and 2% for 13min. Up to 15 of the most intense peptide ions (>5000 counts) were selected and fragmented by MS/MS using multistage activation in the linear ion trap.Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was performed using an LTQ-Orbitrap Elite (Thermo Fisher Scientific) coupled with an Easy nano-LC 1000. The LC solvents were 0.1% formic acid in HRaw data were processed by Proteome Discoverer software  and searched against the Arabidopsis protein sequence database  using an in-house Mascot server . The following parameters were specified in the protein database searches: only tryptic peptides with up to two missed cleavage sites were allowed as done previously (http://phosphat.uni-hohenheim.de/)  and 8–14 . As stages 1–3 anthers are too small to dissect from the floral buds, we collected anthers at stages 4–7 (phase I) for the first sample group. In addition, we sampled anthers at stages 8–12 , as the late phase of anther development.Supplementary Table S1 at JXB online), which corresponded to 5380 proteins  compared with the whole Arabidopsis proteome .Proteomic and phosphoproteomic analyses were performed in parallel for the two samples. One-tenth of the protein extracts was used to determine the protein expression patterns, while the remaining nine-tenths was used to characterize the protein phosphorylation profiles. In total, we identified 20 815 tryptic peptides  containing 3908 phosphorylation sites . Because the phosphopeptides were enriched prior to MS analysis, it was not possible to estimate the fraction of a protein being phosphorylated. A comparison of the phosphorylation sites and their corresponding phosphoproteins identified in the anthers from two phases showed that 646 phosphoproteins and 926 phosphorylation sites were identified in both samples; 870 and 276 phosphoproteins as well as 2006 and 976 phosphorylation sites were uniquely identified in the samples from phases I and II, respectively . An extraordinarily large number (46) of phosphorylation sites were identified for AT5G07530.1, an oleosin domain-containing glycine-rich protein, in phase II. It is expressed specifically during flower stages 10–12 and deposited on the pollen wall (Supplementary Table S3), suggesting extensive regulation of the transcriptional networks by protein phosphorylation.The number of phosphorylation sites in a specific phosphoprotein varied considerably in both samples. Although most proteins had only a few detected phosphorylated sites, a number of proteins were identified with more than 15 phosphorylation sites . These findings are consistent with previous results in mammals and plants  and 89.59% (1704/1902) were on serine (pSer), 9.55% (280/2932) and 10.41% (198/1902) on threonine (pThr), and 0.89% (35/2932) and 0% on tyrosine (pTyr), respectively . After phosphopeptide enrichment, 806 and 569 additional proteins were identified in anther phases I and II, respectively. According to the exponentially modified protein abundance index (emPAI) value, which is commonly used to estimate protein abundance from large-scale identification data by LC-MS/MS, provided by the Mascot search engine  are not abundant analyses . Furtheranalyses . TherefoSupplementary Fig. S4). On the other hand, the regulatory pathways were greatly under-represented in both anther phases, especially in anther phase II . However, Mapman analyses showed opposite results for the phosphoproteomic datasets; the regulatory pathways were highly over-represented in both anther phases  to retrieve the domain information. A comparison of the positions of the identified phosphorylation sites with the positions of functional domains predicated by Pfam analysis showed that 74.76% and 79.57% of the phosphorylation sites were localized outside the functional domain in phases I and II, respectively, whereas only 25.23% and 20.4% of them were located inside functional domains  and (2) with eight angiosperm species . Then, the amino acid similarity was calculated according to the BLOSUM62 scoring system . Among them, 39 were phosphoSer motifs and six were phosphoThr motifs.Reversible protein phosphorylation is mediated by protein kinases and phosphatases, and different kinases preferentially phosphorylate specific substrates with conserved sequence motifs. To predict the relationship between the phosphorylation sites and the corresponding kinases during different anther phases, we performed the motif analysis using Motif-X to extract the over-represented patterns of amino acids from the input sequences containing phosphorylation sites . In totaPlant casein kinase II (CKII), a ubiquitous Ser/Thr kinase localized in nuclear or cytoplasmic compartments, is involved in numerous biological processes during plant growth and development and mainly targets nuclear proteins, notably TFs . Anther development is controlled by a complex transcriptional regulatory network including numerous key TFs (Supplementary Table S5). In anther phase I, the percentages of both TRs and TFs in the phosphoproteomic dataset were more than two times higher than that in our proteomic dataset and the predicted Arabidopsis proteome. However, in phase II, the percentages of identified TRs and TFs among phosphoproteins were two times higher than that among the identified proteins from proteomic analysis, but only slightly higher than that in the Arabidopsis proteome (Transcription regulators (TRs) modulate transcription either as TFs or regulating the activities of TFs. There are 2286 Arabidopsis TFs in the Plant Transcription Factor Database (proteome .P<0.05) in our results. Members of three TF families were only detected in proteomic datasets, including Alfin-like, Nin-like and Whirly families; especially the Nin-like family was only detected in phase II. Three members in the BES1 family  were phosphorylated and detected in anther phase I but not phase II. Members of bZIP family were identified only as the phosphorylated form in both anther phases, suggesting that phosphorylation may play crucial roles in regulating the activities of bZIP TFs during anther development. The CAMTA family TFs were only observed in anther phase I, mainly in phosphorylated form, suggesting that phosphorylation may affect their activities.To assess whether there is a difference in phosphorylation on TF families in different anther phases, we analysed TF families for enrichment using a one-side Fisher’s exact test of the hypergeometric distribution of the targeted sets (43 TF families from our results) compared with the background set (65 families in the Arabidopsis proteome) for identified proteins and phosphoproteins from the two phases. As shown in http://bioinformatics.cau.edu.cn/athKD/index.htm). Kinases are usually regulated by phosphorylation, either through autophosphorylation or by other kinases (Protein kinases and phosphatases play important roles in plant development (Supplementary Table S6). Six kinase and four phosphatase families were enriched from our proteomic and phosphoproteomic datasets. The dual specific protein phosphatase family was observed only in the phosphoproteomic dataset of anther phase I, indicating the abundance of proteins in this family is low and phosphorylation might regulate their activities in anther phase I. Phosphatase and tensin homologs were detected in both anther phases but only in phosphorylated forms, suggesting that phosphorylation may affect their activities in both anther phases. Members of GmPK6/ArMRK1, IRE/NPH/PI dependent/S6 kinase and STE20-PAK like protein kinase families were also detected in both phases, while only the phosphorylated form was observed in phase I. Most members of the other five enriched families were detected in both proteomic and phosphoproteomic datasets from both phases.To estimate the phosphorylation extent of kinase families regulating the signal pathways during anther development, a kinase family enrichment analysis was performed on the identified protein datasets (59 kinase and phosphatase families from our results) compared with the background dataset (91 families in the Arabidopsis proteome) (Supplementary Table S7). Further analysis revealed that one ROS producer  and four ROS scavenging enzymes (two thioredoxins (TRXs) and two glutaredoxins (GRXs)) were phosphorylated in the phase II, while none of the ROS-related proteins was phosphorylated in phase I.Redox state, indicating the level of reactive oxygen species (ROS), was proposed as an important factor for specifying male germ cell fate in plants . In addi2+ binding synergistically activate the ROS-producing enzyme activity of RBOHD in Arabidopsis  family and the Arabidopsis genome encodes ten RBOH family members. Phosphorylation and Cabidopsis . TRXs cobidopsis . Two Arabidopsis . A cystaSupplementary Table S8). The negative regulators of the BR signaling pathway, a GSK3 family kinase BIN2 (Brassinosteroids (BRs) regulate many processes of plant development, e.g. cell elongation, cell division, flowering time control, and responses to biotic and abiotic stresses . Recentlase BIN2 , three B3DB database (Supplementary Table S9). The previous datasets were generated from large-scale phosphoproteomic studies of tissues other than anthers, even not part of the inflorescence, suggesting that the 493 phosphoproteins might be largely specifically expressed or phosphorylated in the anther or other floral organs. To test this idea, we further evaluated the expression of genes for the 493 phosphoproteins in different Arabidopsis tissues . This provides evidence that protein phosphorylation regulates other processes from the transcriptional to post-translational levels. Further comparative analysis of transcriptomic datasets on mutants of ASK1, encoding a ubiquitin-dependent protein kinase, and SDS, encoding a meiotic cyclin-like protein (ask1 mutant (in press) and our phosphoproteomic results showed that protein products of numerous up/down-regulated genes were phosphorylated. This suggests phosphorylation may regulate or coordinate other post-translational modifications during anther development.In our phosphoproteomic analysis, we found that numerous proteins involved in other post-translational modifications were phosphorylated. For instance, there were 15 phosphoproteins involved in acetylation/deacetylation, ten in DNA/protein methylation, four in protein sumoylation and 32 in ubiquitination (S11). Interestingly, the LRR-receptor-like kinase BAM1 was phosphorylated in both phases , whereas phosphorylated BAM2 (at S517) was only detected in phase II. It has been reported that BAM1 and BAM2 together regulate the specification of somatic cells during early anther development (BAM1 is mutated) in early anther development. In a similar situation, MAPK6 was also phosphorylated in both phases (at T221 and Y223), whereas ER was only phosphorylated in phase I (at S975), indicating that MAPK6 may play important roles throughout anther development while ER primarily functions only during early anther development . Phosphorylation of SCC3 and RBR1 were detected in both phases. Six known meiotic proteins were solely phosphorylated in phase I, and these are important for chromosome condensation (CHR4), DNA double-strand break repair , crossover formation (RFC1) and chromosome segregation (WAPL1). A member of the mei2-like gene family, AML5, is involved in formation of SPO11-1-independent chromosome fragmentation and was phosphorylated in phase II, after meiosis is completed. This suggests that either phosphorylation negatively regulates its activity or it may have functions other than in meiosis during late anther development.Meiosis is essential for the formation of functional gametes and previous studies found that protein phosphorylation plays crucial roles in animal meiosis . In our Supplementary Table S12). Interestingly, LRR-RLK family members were only phosphorylated in the anther, consistent with the previous findings that several members of the LRR-RLK family, e.g. EMS1, BAM1/2, SERK1/2 and ER/ERL1/ERL2, play critical roles in early anther development. CRPK-RLK and RLCK VII subfamilies were phosphorylated in mature pollen, which is supported by genetic evidence that CRPK-RLK family members FERRINIA, THESEUS1 and ANX1/2 are preferentially expressed in pollen and regulate pollen tube growth  kinase families identified in two phases and mature pollen .To investigate the possible regulatory network mediated by protein phosphorylation, we predicted the kinase–substrate relationships according to the results from motif analysis and the PhosPhAt database . This is consistent with essential roles of MAPK6 in anther development . Only functional bins with P<1.0×10–2 are presented.Figure S2. Overlap of proteomic and phosphoproteomic analyses in anther phase I and II.Figure S3. The enriched MapMan bins (bin numbers and bin names) of identified phosphoproteins  for anther phase I and II. Only functional bins with P<1.0×10–2 for identified proteins are presented.Figure S4. The enriched MapMan bins (bin numbers and bin names) of proteomic datasets for anther phase I and II. Only functional bins with P<1.0×10–2 for identified proteins are presented.Figure S5. Phosphorylation motifs of phosphoSer and phosphoThr predicted by Motif-X.Figure S6. The kinase–substrate network extracted from phosphoproteome of anther phase II.Table S1. Identified total peptides and phosphopeptides in proteomic and phosphoproteomic analyses of anther phase I and II.Table S2. Identified proteins (including phosphoproteins) in anther phase I and II.Table S3. Overview of phosphoproteins identified in anther developing phase I and II.Table S4. Phosphorylation motifs enriched by motif-X and putative protein kinase or binding proteins.Table S5. Overview of identified transcription regulators in proteomic and phosphoproteomic analyses of anther phase I and II.Table S6. Overview of identified receptor like kinases in proteomic and phosphoproteomic analyses of developing anther and pollen.Table S7. Identified ROS related proteins in proteome and phosphoproteome analyses of developing anthers.Table S8. Identified BR signal proteins in anther phase I and II.Table S9. Overview of 493 novel phosphoproteins.Table S10. Overview of identified anther development and meiosis key proteins.Table S11. Comparison of phosphorylated kinase between developing anthers and mature pollens.Table S12. Identified phosphoproteins involved in other protein/DNA modificationsTable S13. List of predicated kinase to substrate relationships in phosphoproteomic analyses of two anther phases.


Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer. Microtubule-targeting agents are used successfully as anticancer therapeutics. Here authors develop a fluorescence-anisotropy-based assay to identify and characterize ligands for the maytansine site of tubulin and provide crystal structures of identified ligands in complex with tubulin. By perturbing microtubule dynamics during mitosis, these drugs interfere with mitotic spindle formation and thus cell division; however, they also act on interphase microtubules and, as a consequence, affect the intracellular trafficking of important molecules and organelles, in particular in neurons3.The αβ-tubulin heterodimer is the building block of microtubules that, together with F-actin and intermediate filaments, constitute the cytoskeleton. As such, tubulin is an important target for antineoplastic drugs like taxol and vinblastine7. Molecules that bind to the taxane and laulimalide/peloruside site stabilize microtubules, while compounds targeting the colchicine, vinblastine, pironetin or maytansine site destabilize microtubules. Taxane- and vinblastine-site ligands are in clinical use for cancer therapy, but no drugs have been approved that target any of the other four binding sites, with the exception of maytansine that is part of an antibody-drug conjugate (ADC). Furthermore, the clinical application of approved MTAs is hampered by their severe toxic side effects and the development of resistance8.Microtubule-targeting agents (MTAs) can be broadly divided into microtubule-stabilizing and -destabilizing agents. Six distinct tubulin-binding sites for ligands have been structurally characterized to date, which are referred to as the taxane, laulimalide/peloruside, colchicine, vinca, pironetin and maytansine site, respectively9. It is a unique site on β-tubulin that is located at the longitudinal tubulin–tubulin interface in microtubules, which readily explains the microtubule-destabilizing effects of maytansine-site ligands. Three distinctly different ligands that target the maytansine site have been described: maytansine, PM060184 and rhizoxin9. Maytansine has been successfully incorporated into the ADC trastuzumab emtansine, which is approved for the treatment of breast cancer10. In principle, ADCs can overcome the toxicity problem associated with MTAs; however, the costs for developing and using ADCs in targeted therapy are a major drawback11 and the use of traditional anti-tubulin agents still remains a valuable approach. PM060184 is in phase II clinical development for the treatment of breast cancers , while rhizoxin had reached phase II, before being discontinued for reasons that are poorly understood12.The maytansine site on tubulin has been discovered only very recentlyIn this study, we develop a quantitative fluorescence anisotropy displacement assay based on a fluorescein-labeled maytansine derivative, with the aim to provide a platform for the identification and characterization of additional maytansine-site ligands. We choose the maytansine site because it is poorly characterized and because no tools are available to characterize the binding of maytansine-site ligands in detail. We show that the assay is specific for the maytansine site and can be operated in a high-throughput manner. By using this assay, we identify two natural products, spongistatin-1 and disorazole Z, as maytansine-site ligands. We solve the structures of both compounds in complex with tubulin to high resolution by X-ray crystallography, which allows us to analyze the maytansine site in great detail. The experimental tools and results presented in this study should contribute to the discovery and characterization of maytansine site-directed, small-molecule MTAs for the development of next-generation anti-tubulin drugs for the treatment of cancer.M5)). In the tubulin–maytansine crystal structure9, the corresponding N-acetyl-N-methyl-d-alanyl residue in maytansine occupies a solvent-exposed position that is located remote from the tubulin surface; thus, the fluorescein-linker moiety was expected not to interfere with binding of the FcMaytansine probe. As shown in Fig. M1). Details of the synthesis can be found in the Supplementary Information.In this study, we sought to develop a fluorescence anisotropy assay to identify and determine the tubulin-binding affinities of maytansine-site ligands. To this end, we prepared a fluorescently labeled maytansinoid that carries a fluorescein reporter attached to the 3-OH group of the maytansinol core structure via a flexible linker moiety  , the addition of increasing amounts of tubulin (0 to 400 nM) to FcMaytansine (10 nM) led to a concentration-dependent increase in anisotropy by a factor of ~1.6 from ~16 mA in the absence of tubulin to ~250 mA at saturation Fig. , thus pr 5) Fig. . A Kd ~o2), the stathmin-like protein RB3 (R) and tubulin tyrosine ligase (TTL)17, and the structure of the T2R–TTL–FcMaytansine complex was solved at 2.4 Å resolution .To demonstrate that FcMaytansine does indeed specifically bind to the maytansine site of tubulin, we used X-ray crystallography. FcMaytansine was soaked into a crystal composed of a complex formed between two αβ-tubulin heterodimers (T19. Vinblastine and colchicine were used as negative controls. The concentrations of FcMaytansine (10 nM) and tubulin (4 nM) were chosen such that the bound fraction of FcMaytansine was around 0.5 based on the Kd of the tubulin–FcMaytansine complex. The mixture was then incubated with an unlabeled competitor at increasing concentrations prior to anisotropy measurements. The final dimethyl sulfoxide (DMSO) concentration in each sample amounted to ~1.2%; as documented in Supplementary Fig. Kd values determined for ansamitocin P3 and PM060184 were 15.0 ± 1.3 nM  and 111 ± 30 nM , respectively.Next, we performed fluorescence anisotropy competition experiments with FcMaytansine and the two unlabeled maytansine-site ligands ansamitocin P3 (a maytansine derivative) and PM060184Z-factor20. Samples of 10 nM FcMaytansine mixed with 400 nM tubulin and of 10 nM FcMaytansine alone were applied to a 96-well plate and the anisotropy was measured , most likely as a consequence of reduced cellular uptake22. At the same time, the intracellular formation of M9 from an anti-EpCAM-DM1 conjugate in MCF-7 cells has been shown to correlate with the effects of the conjugate on microtubule dynamicity23. While this finding indicates that the cytotoxicity of the anti-EpCAM-DM1 conjugate is based on M9-mediated suppression of microtubule dynamics, to the best of our knowledge, a direct interaction of M9 with tubulin has not been demonstrated. Using our fluorescence anisotropy displacement assay, we have thus determined the binding affinity of M9 for tubulin.The maytansine derivative DM1 Fig.  forms th M9 Fig.  as the pM9 . The reaction produced two high-performance liquid chromatography (HPLC)-separable, but slowly interconverting, C9’-diastereoisomers (designated as M9A and M9B) in an approximately 1:1 ratio, with full equilibration in phosphate buffer pH 7.2 being observed after 24 h. This finding is in line with previous data suggesting that the thiol addition products of N-ethyl maleimide with different biological thiols (including cysteine and glutathione) readily interconvert even at neutral pH24. The method used to prepare M9 inevitably leads to two diastereoisomeric products, due to the formation of an additional stereocenter at C9’ in the 1,4-addition of DM1 to the maleimido part of the linker precursor and the inherently non-selective nature of the reaction. However, the interconversion rate between M9A and M9B is sufficiently slow to allow the separate assessment of their tubulin-binding affinities. As shown in Fig. Kd values of 12.6 ± 0.4 nM  and 17.5 ± 1.2 nM  for M9A and M9B, respectively, values similar to the one obtained for the maytansine derivative ansamitocin P3. These Kd values are in the same ballpark as the one obtained for FcMaytansine using direct titration experiments (see above), indicating that the fluorescein moiety does not significantly influence the affinity of FcMaytansine for tubulin.The DM1-lysine conjugate M9 Fig.  was prepM9 is very similar to that of S-Me DM1, which suggests that the difference in antiproliferative activity when the compounds are added to cells exogenously is due to differences in cell uptake. In contrast, the potency of intracellularly generated M9 should be comparable with that of maytansine .Together, these data demonstrate that the affinity of 27. This misassignment can be explained by the fact that the maytansine and vinblastine sites are located close to each other and, thus their distinction is not straightforward    and rhizoxin had been previously thought to bind to the vinblastine site of tubulin 3) Fig. . Likewis 3) Fig. .Fig. 4Sp2R–TTL crystal system and solved the tubulin–spongistatin-1 and tubulin–disorazole Z complex structures to 2.4 and 2.1 Å resolution, respectively , a hydrophobic contact point IB (mediated by βTrp407) and a hydrophobic contact point IC (mediated by Phe404), respectively. Furthermore, PM060184 and spongistatin-1 both interact with two pockets adjacent to the common pharmacophore point II via hydrogen bonds  and hydrophobic contacts . Interestingly, spongistatin-1 establishes two distinct interactions close to point II via a hydrophobic contact  and a weak hydrogen bond , thereby defining an additional binding mode that is not observed for any of the other four maytansine-site ligands. Overall, this analysis provides a comprehensive and detailed comparison of the tubulin-binding modes of maytansine-site ligands.Collectively, the crystal structures of tubulin in complex with spongistatin-1 and disorazole Z together with those of the complexes with maytansine, PM060184, and rhizoxin Fnds Fig. . MaytansKd values in the nanomolar range in a fast, cheap, non-destructive and easily automatable manner. Using this assay, we determined the dissociation constants of a number of important maytansine-site ligands, including the pharmacologically active entity of the ADC trastuzumab emtansine and the phase II compound PM060184. We further identified spongistatin-1 and disorazole Z as canonical maytansine-site ligands and we have structurally characterized the maytansine site in great detail.In this study, we have established a high-performance assay that allows the identification and quantitative evaluation of ligands that specifically bind to the maytansine site of tubulin. Our FcMaytansine probe enables the screening of ligands with Our results have several implications. Firstly, our experimental platform will allow the curation of assignments of ligands that may be thought to bind to the vinblastine site erroneously. We expect that the number of ligands binding to the maytansine site will further increase and establish it as a major ligand binding site on tubulin along with the taxane, colchicine and vinblastine sites. Secondly, our FcMaytansine approach paves the way to the generation of additional tool compounds to be used in in vitro and in cellular experiments. The maytansine site of β-tubulin is exposed at the plus-end of microtubules. Exchanging the fluorescein moiety of FcMaytansine by a stronger fluorophore could thus be used to visualize distal microtubule plus-ends by fluorescence microscopy and study the action of single drug molecules on their molecular target. Thirdly, maytansine derivatives bearing fluorescent groups other than fluorescein could be used to develop assays for the identification of low micromolar affinity binders, as they are typically found in small molecule and fragment libraries. To the best of our knowledge, there are no structurally simple , potent maytansine-site lead compounds available that could potentially be developed into (free) drugs for clinical use. On the other hand, the detailed structural description of the maytansine site may now enable the rational design of such less complex maytansine-site-directed MTAs. Overall, our study presents a possible basis for the development of next-generation anti-tubulin drugs for the treatment of cancer.M5 and the DM1-lysine conjugate M9 are reported in the Supplementary Information . Ansamitocin P3, colchicine and vinblastine were obtained from Sigma Aldrich. Maytansinol was purchased from Levena Biopharm, San Diego, USA.Details of the synthesis of the fluorescent maytansinoid 2 and 0.2 mM EDTA) was titrated with increasing amounts of tubulin up to 400 nM. The anisotropy and fluorescence values of the free ) and bound ) states were determined in the absence and presence of 400 nM tubulin, respectively. The anisotropy was measured using a PHERAstar microplate reader (BMG Labtech) with the fluorescence polarization module. Samples were equilibrated at 25 °C for 15 min prior to measurement at 25 °C. Excitation and emission wavelengths were set to 485 and 528 nm, respectively. The read height and the gain were adjusted automatically. As a canonical maytansine-site compound we used ansamitocin P3, a close derivative of the parent compound maytansine. Comparison of the high-resolution tubulin-ansamitocin P3 crystal structure (our own unpublished results) with that of tubulin–maytansine9 showed that all tubulin-contacting groups of the two compounds are identical. We thus consider ansamitocin P3 in this context as an excellent substitute for maytansine.The binding constant of the fluorescent FcMaytansine probe to tubulin was determined by fluorescence anisotropy titration at 25 °C in black Greiner 384-well, flat-bottom microplates and in a final volume of 50 µL. 10 nM FcMaytansine in assay buffer  and free (νf) fluorophore coincides with the fluorescence fractions for the bound and free fluorophore . Employing Eq. In the simplest case of a fluorescent probe with no quantum yield difference between the bound and the free stateHowever, FcMaytansine does not exhibit this behavior, because there is a 3.1 ± 0.1-fold difference between the fluorescence intensity of the bound and free FcMaytansine. In this case the fluorescence fraction is weighted by the change in fluorescence intensity according toR representing the ratio between the fluorescence intensity of the bound and free species (R = Ib/If). Equation R = 1)36.Employing Eqs. 8 M−1 using SigmaPlot v13 .Once the fractional saturation of FcMaytansine in the experiments is known, the free concentration of tubulin and FcMaytansine in the titration assay can be calculated from the difference between the bound and the total concentration of the probe. The free concentration of tubulin was calculated from the difference in the total and bound concentration of FcMaytansine assuming a stoichiometry of 1:1. The values of fractional saturation of FcMaytansine bound vs. the free tubulin concentration were used to determine a binding constant at 25 °C of 1.6 ± 0.3 × 10l) can be determined from the known values of the binding constant of the reference ligand K(r) and the total concentrations of binding sites, FcMaytansine and problem ligand by solving the simultaneous mass action equations:The binding constant of a problem ligand K.The system was solved using EQUIGRA52R–TTL were obtained by the sitting-drop vapor-diffusion method as previously described17 and with a protein ratio of 2:1.2:1.2 (tubulin/RB3/TTL). Crystals reached their maximum dimensions within 1 week and were soaked for 1 h to overnight at 20 °C in reservoir solution consisting of 10 % PEG 4k, 16% glycerol, 30 mM MgCl2, 30 mM CaCl2 and 100 mM 2-(N-morpholino)ethanesulfonic acid/imidazole pH 6.7 and containing 2 or 5 mM of compound . Crystals were fished directly from the drop, transferred for a few seconds to a reservoir solution supplemented with 2 or 5 mM of compound and 20% glycerol as cryoprotecting agent and flash-cooled in a nitrogen stream at the beamline. Standard data collections were performed at 1 Å wavelength at 100 K at the beamline X06DA at the Swiss Light Source . Standard data processing, structure solution using the difference Fourier method and structure refinement were performed with the programs XDS37 and Phenix38,17. All models have good geometry, with small r.m.s. deviations from ideal values for bond lengths and bond angles. MolProbity39 analysis shows all residues in favored or allowed regions in the Ramachandran plot . Data collection and refinement statistics are given in Table Crystals of Twww.rcsb.org) under accession numbers 6FJF (tubulin-FcMaytansine), 6FII (tubulin-spongistatin-1) and 6FJM (tubulin-disorazole Z).Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. Coordinates of the X-ray crystal structures have been deposited in the RCSB PDB (Supplemental InformationPeer Review File