Plasmodium falciparum, is able to evade spleen-mediated clearing from blood stream by sequestering in peripheral organs. This is due to the adhesive properties conferred by the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family exported by the parasite to the surface of infected erythrocytes. Expression of the VAR2CSA variant of PfEMP1 leads to pregnancy-associated malaria, which occurs when infected erythrocytes massively sequester in the placenta by binding to low-sulfated Chondroitin Sulfate A (CSA) present in the intervillous spaces. VAR2CSA is a 350 kDa protein that carries six Duffy-Binding Like (DBL) domains, one Cysteine-rich Inter-Domain Regions (CIDR) and several inter-domain regions. In the present paper, we report for the first time the crystal structure at 2.9 Å of a VAR2CSA double domain, DBL3X-DBL4ε, from the FCR3 strain. DBL3X and DBL4ε share a large contact interface formed by residues that are invariant or highly conserved in VAR2CSA variants, which suggests that these two central DBL domains (DBL3X-DBL4ε) contribute significantly to the structuring of the functional VAR2CSA extracellular region. We have also examined the antigenicity of peptides corresponding to exposed loop regions of the DBL4ε structure.The human malaria parasite,  Plasmodium falciparum malaria arise from sequestration of parasitized erythrocytes (PEs) in diverse tissues, such as the microvasculature of different organs or the intervillous spaces of the placenta, as well as by adhesion to host cells, such as non-infected erythrocytes and plateletsP. falciparum erythrocyte membrane protein (PfEMP1) adhesin family, which is encoded by a family of roughly 60 var genesMost clinical manifestations of via attachment to the glycosaminoglycan chondroitin sulphate A (CSA) present in the intervillous spaces5var2csa is the only var gene transcribed in placental isolates or CSA-binding laboratory strains and disruption of var2csa leads to the irreversible loss of CSA-binding phenotype791011Pregnancy-associated malaria (PAM) results from the accumulation of PEs in the placenta The extracellular region of VAR2CSA comprises six DBL domains (type ε or unknown (X)) and a CIDR (CIDRpam) module arranged in the following configuration5Although single recombinant domains have been shown to bind CSA161719192425We have embarked on a structural study of VAR2CSA multidomain constructs in order to analyze the structural organization of the domains in detail. Here we report the crystal structure of the DBL3X-DBL4ε double domain from the FCR3 strain. The structure of the FCR3-DBL3X domain has already been described242 is buried at the interface between DBL3X and DBL4ε.Expression of the recombinant FCR3-DBL3X-DBL4ε double domain has been previously describedThe structure of the FCR3-DBL3X single domain has been previously published2410H9 and αH9.The structure of DBL4ε, the least polymorphic VAR2CSA domain, has not been previously described; however, the pattern of DBL secondary structure is generally preserved . As in oAs our study provided the first experimental structure of the DBL4ε VAR2CSA module, we examined the structural context of antigenic regions in this domain. Accordingly, we assessed the antibody response of a rabbit, immunized with the DBL3X-DBL4ε double domain, to a selected set of DBL4ε peptides. We selected peptide sequences corresponding to exposed loop regions that connect the conserved helices of the DBL fold. Some segments in Subdomain 3, however, were excluded since certain regions could not be traced in the electron density maps or because the presence of several cysteine bridges might compromise structural mimicry of these regions by peptides. Using these criteria, we selected six polypeptide segments denoted Pep-1 to Pep-6, which are detailed in Antigenicity of the peptides was examined with protein G-purified IgG from a rabbit immunized with the recombinant FCR3-DBL3X-DBL4ε protein. Two different experimental protocols were followed to assess consistency of the results. The first approach used solid phase ELISA, where the peptides were coated onto 96-well plates and reacted with the purified IgG . Pre-immIn the second experimental protocol, peptide-specific antibodies were purified by binding the immune IgG to the peptides immobilized on streptavidin-coated magnetic beads. After elution from the beads, antibodies from each of the peptides were tested in turn by ELISA for reactivity with the FCR3-DBL3X-DBL4ε immunogen, as well as with 3D7-DBL1X-DBL2X and Bovine Serum Albumin negative controls . ConsistWe therefore conclude that peptides Pep-2, Pep-3, Pep-4 Pep-5 and Pep-6 are antigenic. More importantly, the fact that immune IgG purified on each of these peptides reacted with the recombinant FCR3-DBL3X-DBL4ε confirms that the corresponding regions on the DBL4ε structure are also antigenic.Our study has aimed to extend structural knowledge of the individual VAR2CSA domains and to contribute to understanding of the interdomain organization of its extracellular region. The structure of the double domain FCR3-DBL3X-DBL4ε provides the first structural description of the DBL4ε module, which is the least polymorphic domain of VAR2CSA, and gives a detailed atomic view of the interdomain interactions between the two central domains of this variant. The double domain forms a compact structure with a short linker of ten residues, K1583 to K1592 (interdomain) region connecting the two DBL modules . The int19The three C-terminal domains of VAR2CSA belong to the ε class of the PfEMP1-DBL fold. The ε class is typified, in particular, by the absence of canonical cysteines Cys(2) and Cys3), which form a disulfide bridge in other DBL classes, and the presence of Cys(10b) and Cys(10c), which form a bridge. Moreover, Cys(10c) always precedes Cys(10a) by two residues, which f26Our study provides the first structure determination of a VAR2CSA DBL4ε domain. The canonical Cys(-1)-Cys(5a) disulfide bridge, linking subdomains 1 and 2 of PfEMP1-DBL domains, is absent in FCR3-DBL4ε structure; this absence is characteristic of the DBL4ε-VAR2CSA domain in other VAR2CSA variants157W, where this helix is kinked at the 4th residue. In DBL4ε-FCR3, however, the interval between the conserved tryptophan residues comprises 18 amino acids (WWX18W), of which 11 form a loop with no regular secondary structure. This loop is surface exposed and does not interact with the DBL3X domain. In different DBL4ε-VAR2CSA domains, the distance between the conserved tryptophan residues varies between 18 to 25 amino acids and the region is highly variable in sequence, except at the N- and C-terminal ends; this would correspond to an insertion of a loop comprising 11 to 18 residues into the αH5 helix in the different VAR2CSA strains. This region has been previously noted as a variable block region unique to the DBL4ε domainIn all known structures of DBL domains, whether from PfEMP1 or erythrocyte-binding proteins , helix αH5 contains a conserved motif WWXSince the structure of DBL5ε has not yet been determined, the discussion is restricted to sequence analysis only. Canonical Cys(5) and Cys(6) are disulfide bridged in the DBL motif; however, while Cys(6) is present in all known DBL5ε-VAR2CSA sequences (C2171 in FCR3), Cys(5) is absent. The loop between Cys(10b) and Cys(10c) is only two residues long and is quite well conserved in sequenceCrystal structures of DBL6ε from the FCR3 and 3D7 laboratory strains have been solved26P. falciparum infected erythrocytes in a strain-independent manner, showing that this domain is surface-exposed in the full-length VAR2CSA2935363821Several studies analysing the immune response to VAR2CSA have demonstrated that DBL4ε-specific antibodies are able to label and inhibit the adhesion of et al.et al.et al. to cover this loop region and used to immunize rats, failed to elicit an efficient immune response against the native VAR2CSA expressed on infected erythrocytes.Our results correlate in part with previous pepscan mapping and B-cell epitope prediction of the FCR3-DBL4ε sequence using rat and human immune antibodiesOur crystallographic study of the FCR3-DBL3X-DBL4ε double domain is a first step towards providing atomic detail of the domain organisation of the extracellular region of VARCSA. It has revealed an interface between the DBL3X and DBL4ε domains that is highly conserved within VAR2CSA variants  and is t19All animal vaccination experiments were executed in strict accordance with good animal practices, following the EU animal welfare legislation and after approval of the Biotem and INSERM ethical committees. Every effort was made to minimize suffering.E. coli SHuffle® strain of (Novagen). Cells were centrifuged and resuspended in lysis buffer  and lysis was performed with an Emulsiflex homogeniser (Avestin). Lysate was cleared by centrifugation at 8000 g during 1 h. FCR3-DBL3X-DBL4ε was first captured on a heparin column and eluted with 20mM Tris-HCl, 1M NaCl, pH8. The eluate was loaded on a metal affinity column  and FCR3-DBL3X-DBL4ε recovered with 20 mM Tris-HCl, 150 mM NaCl, 200 mM Imidazole, pH8. A final purification step to remove possible impurities and aggregates was carried out by gel filtration . In order to improve crystal quality, the protein was subsequently subjected to an additional purification step on a CM-Sepharose column  equilibrated in 20 mM HEPES pH 7, 40 mM NaCl. The protein was eluted by increasing NaCl concentration by 5% (w/v) steps and concentrated to 9 mg/ml.Expression of the recombinant FCR3-DBL3X-DBL4ε double domain has been previously describedCrystals were grown using the hanging-drop vapour-diffusion technique by mixing 1 μL protein solution with 0.75 or 1 μL reservoir solution and equilibrating against 1 mL reservoir solution. Initial crystallisation conditions were identified using robotic screening with several commercial crystallisation screens at the Crystallography Platform of the Pasteur Institute. These conditions were further refined by manual experiments using the PEG-Ion screen (Hampton). The best crystals were grown in 18% PEG 3000, 100 mM Bis-Tris buffer, pH 6.5, 300 mM NaCl and 10% v/v glycerol for the first data-quality crystal, none for the second one. Crystals were mounted on cryoloops, rapidly passed through 15% glycerol and flash-frozen in liquid nitrogen for data collection.Native data were collected on the ID29 beamline at the ESRF, Grenoble, France and on Proxima1 at SOLEIL, St. Aubin, France. All data were treated with XDS44. Although the diffraction was weak, we found it important to include intensities to 2.9 Å resolution. As has been underlined elsewhereThe structure determination was initiated with molecular replacement using AMoReThe anti-FCR3-DBL3X-DBL4ε rabbit IgGs used in this study were produced by immunization of a New Zealand white rabbit. Immunization with the recombinant double domain was performed by Biotem, France, according to animal immunization guidelines. In brief, a NZ white rabbit received intradermally 40 μg of recombinant protein in Freund’s Complete Adjuvant for first immunization and 20 μg subcutaneously for three boosts. Antisera were collected 63 days after the first injection. IgG were purified from sera by affinity chromatography on protein G.via a linker comprising ε-aminohexanoic acid (Ahx) and two glycine residues. This allowed fixing a constant quantity of each peptide to streptavidin-coated beads and minimized potential steric hindrance, favouring more efficient antibody recognition. A quantity of 2.6 × 10−8 mol of each biotinylated peptide was incubated with 200 μL streptavidin-coated magnetic beads  and protein G-purified antibodies from a rabbit immunized with FCR3-DBL3X-DBL4ε were affinity purified on these peptides. 250 μg of immune or pre-immune protein G-purified antibodies diluted in PBS were incubated with 200μL beads (capacity 0.4 × 10−9 mol of peptides/mL), pre-equilibrated with PBS. After extensive washing of the beads with PBS, antibodies bound to the beads were eluted with 100 μL elution buffer . The eluate was neutralized with 20 μL of neutralizing buffer .Peptides corresponding to exposed loop regions of the DBL4ε domain  were proPeptide-purified IgG was tested either on the recombinant domain or on the biotinylated peptides. ELISA was performed on recombinant proteins as follows. Recombinant FCR3-DBL3X-DBL4ε or 3D7-DBL1X-DBL2X (used as a negative control) was coated overnight at 4 °C on 96-well plates at a concentration of 1 μg/mL. Wells were coated with blocking buffer  during 1 h at 37 °C. After three further washing steps, wells were incubated with peptide-purified IgG diluted in blocking buffer (1:100) for 1 h at room temperature. IgG were revealed with horseradish-peroxidase-conjugated Ig anti-rabbit IgG (1:200 in blocking buffer), using TMB as a substrate. ELISA was performed on immobilized peptides as follows. Biotinylated peptides were immobilized on 96-well plates at 200 μg/mL in 50 mM carbonate buffer pH 9.6, overnight at 4 °C. After a blocking step, affinity-purified antibodies (dilution 1:100) or an anti-biotin Mouse monoclonal antibody  were added and incubated for 1 h at 37 °C. After extensive washing, horseradish-peroxidase-conjugated Ig anti-rabbit IgG (1:2000) was incubated 1 h at room temperature. TMB was used as a substrate and absorbance measured at 655 nm.How to cite this article: Gangnard, S. et al. Structure of the DBL3X-DBL4ε region of the VAR2CSA placental malaria vaccine candidate: insight into DBL domain interactions. Sci. Rep.5, 14868; doi: 10.1038/srep14868 (2015).


The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk. The Src family of tyrosine kinases (SFKs) constitutes a central hub for cellular signaling and regulation. These kinases phosphorylate many protein substrates associated with cell growth, differentiation, adhesion, motility and invasion –4. Thus,The C-terminal Src Kinase (Csk) serves as the master regulator and suppressor of all SFKs whose mis-regulation leads canonically to tumorigenesis . Csk phocat/Km for Src) by 30-fold, removal of the SH3 domain lowers activity by 11-fold ATP (4000–6000 cpm pmol-1) and 10 mM DTT at lab temperature (23°C). Reactions were initiated by adding ATP to the master mix containing all enzymes and reagents. a fraction of the reaction was quenched at several time intervals and subjected to SDS-PAGE. Bands corresponding to the phosphorylated kdSrc substrate were excised and quantified on the 32P channel in liquid scintillator. The specific activity of [γ-32P]ATP was determined by measuring the total counts of the reaction mixture. The time-dependent concentration of 32P-kdSrc was then determined by considering the total counts per minute (CPM), the specific activity of the reaction mixture, and the background phosphorylation. Non-linear regression analysis were performed to quantify the activity of each construct from phosphorylation time course of a fixed amount of kdSrc. The data were fitted to the following equation to obtain rates : P = A(1-ekt) where P is the phospho-product, A is the total substrate concentration, k is the rate constant, and t is time. Initial velocity experiments were typically initiated with variable amounts of substrates (kdSrc or ATP) and data were fitted to a Michaelis–Menten model to obtain steady state parameters.Time-dependent phosphorylation of kinase dead Src (kdSrc) by wild type or variant Csk enzymes was carried out in assay buffer: 100 mM MOPS (pH 7.0), 100 mM NaCl, 10 mM MgClOptimal proteolysis conditions for CSK were previously established .The instrument setup and operation was previously described . All fro2O of 83%. The deuterated exchange buffer  was prepared using 99.9% D2O and adjusted to pD 7.0 with DCl. 24 μL of the exchange reaction was quenched at specified time points  into pre-chilled high recovery autosampler vials containing 6 μL of quench buffer . The vials were sealed and frozen over dry ice, then stored at -80°C until analysis. The in-exchange control consisted of the protein added directly to the pre-chilled deuterated and quench buffers, then immediately followed by the normal sample preparation procedure. The back exchange control was performed by incubating samples in 0.5% formic acid in D2O buffer overnight.The exchange time course experiments for wild type and variant Csk samples were all performed simultaneously at 23°C as follows: Deuteration was initiated by adding 24 μL of ~20 μM protein stock solution  to 120 μL of the equivalent deuterated exchange buffer for a final DThe identity of the parent peptide ions was determined using the Proteome Discoverer software program (ThermoFisher) and data dependent MS/MS data. The quality of each peptide was monitored by individually examining each measured isotopic envelope spectrum for the entire time course exchange. The deuterium content was calculated for each time point by using specialized software as previously described .The PDB structure 1K9A was prepared using Schrodinger’s protein preparation wizard, modeling in residues of the N-terminus and the activation loop which are not observed in the X-ray structure. The structure was minimized and solvated in an octahedral TIP3P water box. The system was then equilibrated and a 50 ns conventional molecular dynamics NVT simulations was run using the AMBER ff99SB-ILDN force field . The staS1 FigThe root mean square deviations (RMSD) for the SH3 domain  and SH2 domain  are plotted as a function of aMD simulation time. The calculated values are relative to the kinase domain throughout the simulation time.(TIF)Click here for additional data file.S2 FigComputed RMSD of the SH3 domain relative to the crystal structure of CSK for the long trajectory (A) and independent 175 ns simulations (sims B-F). The same transition seen in the long simulation is also observed in shorter simulation (E).(TIFF)Click here for additional data file.S3 FigFrames extracted from two independent simulations are clustered into two states based on the RMSD of the SH3 domain. The fractional occupancy of contacts present in the two states are compared for two simulations  in which the SH3 domain is designated as "up, state 0” or "down, state 1”. Similar occupancy changes are observed for many contacts between the two states indicating that both simulations report on the same transition of the SH3 domain.(TIF)Click here for additional data file.S1 FileFrames extracted from the long and short aMD trajectories are used to visualize the motions of regulatory domains in Csk. Similar motions can be observed in the the long  and the shorter  aMD simulations.(MPG)Click here for additional data file.


Escherichia coli and many other bacteria. The phosphate signal is mediated by a classic two component signal system composed of PhoR and PhoB. The PhoR histidine kinase is responsible for phosphorylating/dephosphorylating the response regulator, PhoB, which controls the expression of genes that aid growth in low phosphate conditions. The mechanism by which PhoR receives a signal of environmental phosphate levels has remained elusive. A transporter complex composed of the PstS, PstC, PstA, and PstB proteins as well as a negative regulator, PhoU, have been implicated in signaling environmental phosphate to PhoR.Proper phosphate signaling is essential for robust growth of This work confirms that PhoU and the PstSCAB complex are necessary for proper signaling of high environmental phosphate. Also, we identify residues important in PhoU/PhoR interaction with genetic analysis. Using protein modeling and docking methods, we show an interaction model that points to a potential mechanism for PhoU mediated signaling to PhoR to modify its activity. This model is tested with direct coupling analysis.These bioinformatics tools, in combination with genetic and biochemical analysis, help to identify and test a model for phosphate signaling and may be applicable to several other systems. For all life, phosphate is an essential nutrient. Bacteria have several mechanisms to scavenge phosphate that are only expressed when the level of available environmental phosphate is limited: including a phosphate specific ABC transporter complex (PstSCAB) and a periplasmic phosphate scavenging enzyme ; the product of the oA gene) . ExpressoA gene) ,3.Escherichia coli, a classic two-component signal transduction system, composed of the PhoR histidine kinase and the PhoB response regulator, is responsible for expression control of a group of genes called the Pho regulon. PhoR consists of an N-terminal membrane-spanning region, as well as cytoplasmic PAS, DHp and CA domains. The PAS domain was named for the Drosophila Per, Arnt, and Sim proteins, in which this domain was originally described and has been found in many signaling proteins. Many PAS domains bind cofactors such as heme [dimerization and contains the site of histidine phosphorylation. The CA domain is the catalytic, ATP-binding part of the protein. PhoB consists of an N-terminal phosphorylation domain that receives a phosphoryl group from PhoR and a C-terminal DNA binding domain. In low phosphate conditions, PhoR acts as a PhoB kinase. Upon phosphorylation, PhoB recruits RNA polymerase to promoters of the Pho regulon that contain a Pho box. In high phosphate conditions, PhoR acts as a phospho-PhoB phosphatase and removes the phosphate from PhoB to keep the expression level of Pho regulon genes very low. One unanswered question with this system is how PhoR perceives external phosphate concentrations. PhoR lacks a significant periplasmic domain that could detect phosphate abundance outside the cell. Past work has shown that the PstSCAB transporter and the PhoU protein play important roles in phosphate signaling to PhoR [In  as heme . The DHp to PhoR . The mecphoU gene leads to poor growth and the frequent development of compensatory mutations in the other Pho regulon expression control genes, pstSCAB, phoR, and phoB [A deletion mutation of the and phoB . The pooand phoB , which land phoB . Referenand phoB  proposedphoU mutants for signaling activity, interactions with PhoR, and interactions with PstB. A scanning mutagenesis screen of the PAS domain of PhoR identified potential residues important for interaction with PhoU. We modeled potential docking interactions between PhoR and PhoU. One of the docking models showed a close proximity of identified residues in PhoU with a predicted interaction loop of the PAS domain of PhoR. To validate this model, we performed a Direct Coupling Analysis (DCA) between PhoR and PhoU using sequences from the gammaproteobacteria. The DCA results are consistent with the proposed docking model and may point to a mechanism of action for PhoU in controlling the opposing kinase and phosphatase activities of PhoR.Recently, PhoU was shown to directly interact with PstB and PhoR . This obphoU gene under salicylate expression control), p116A147E, p116U2 A147K, p116U2 R148A, p116U2 R148E, p116U2 D150A /(4 × OD600 of 1:4 dilution of the overnight culture). Cultures were grown in triplicate and the average is reported with error bars representing the standard deviation.We based our Alkaline Phosphatase (AP) Assays on a 96 well plate β-Galactosidase Activity assay previously described . CultureBACTH analysis and β-Galactosidase Assays were performed as described in . BrieflyE. coli PhoR  [We used the ClusPro webserver -15 to dos mutans ), and Phima PhoU ) using P(Phyre2) .http://www.kegg.jp/). We used a list of PhoR and PhoB orthologs to identify species where both annotated PhoR PhoB encoding genes were found on the chromosome. The PhoR and PhoU sequences were collected manually using the predicated protein domains and the genomic context of the gene to select genes that were most likely correctly annotated. For example, for this analysis we were not interested in histidine kinases annotated as PhoR from species that did not have a phoU gene on the chromosome. Sequences were concatenated starting with PhoU, followed by PhoR from the same species. Twenty alanine residues were artificially placed between the two protein sequences to aid in differentiating the PhoU from the PhoR after the alignment. Using MAFFT version 7 , we were able to create an alignment file to use in DCA analysis. A MATLAB implementation of direct coupling analysis reported in [We used PhoU and PhoR sequences from 196 species of bacteria from the gammaproteobacteria group. Sequences for proteins were collected using the Kegg webserver  [phoU gene into the compatible pKG116 plasmid (CamR) [phoU expression is necessary but not sufficient for proper phosphate signaling and are consistent with a model that these proteins act together in signal transduction.Mutations in any of the d (AmpR)  and the d (CamR) . ControlE. coli isolated mutants that constitutively expressed AP [phoU35 because it was independent of the phosphate transport genes [pstSCABphoU operon were sequenced and the phoU35 mutant was analyzed, they found this mutation encoded a change from alanine at position 147 to glutamic acid (A147E) [phoU deletion mutation results in loss of phosphate signaling and causes a severe growth phenotype, but the phoU35 allele was only reported to cause a loss of signaling without the accompanying poor growth, we hypothesized that the phoU35 mutation may disrupt PhoU's interaction with PhoR, preventing the signal for the switch to PhoR phosphatase activity, but may maintain the interaction with PstB, limiting excess transport of phosphate into the cell during phosphate replete conditions.Early studies of phosphate homeostasis in essed AP . One mutessed AP  was charessed AP  and was rt genes . When th (A147E) . Since aphoU35 allele do not show a severe growth phenotype. We employed a phoU deletion strain in which the normal pstS promoter was replaced by the tac promoter to uncouple expression of this operon from mutations in any of its genes (STACΔphoU) [phoU still in the chromosome grew normally. We observed a significant growth defect when the phoU knockout strain containing an empty vector, pKG116, was grown in identical conditions. When the p116U2 plasmid was introduced into this strain, expressing wild-type phoU, and grown under identical conditions, no growth defect was observed. Moreover, when the phoUA147E allele was introduced into the strain, the growth phenotype was similar to that of wild-type phoU. These results confirm the lack of growth defect in the phoU35 mutant and are consistent with the hypothesis that the A147 residue of PhoU is important for interactions with PhoR.We wanted to confirm that strains expressing the ACΔphoU) . By growphoU35 allele caused a signaling defect when expressed from a multicopy plasmid, we tested AP expression in the strains constructed for the previous experiments. Figure phoU protein in the vicinity of A147 would also display a signaling defect. We therefore introduced additional mutations by site-directed mutagenesis in the region near A147  to create versions of PhoU with different charges and sizes of amino acid side chains in this region. The mutations that we created were A147K, R148E, R148A, and D150A. The rightmost four bars in Figure To verify that the phoU mutant that can interact with PstB to limit phosphate transport but can no longer interact with PhoR to signal the phosphate level.The combination of no growth inhibition and disrupted signaling are the expected phenotypes of a phoU mutant that can interact with PstB to limit phosphate transport but can no longer interact with PhoR to signal the phosphate level. We further examined this hypothesis by using a bacterial adenylate cyclase two-hybrid (BACTH) system to test for interactions between mutant versions of PhoU and PstB and PhoR  and the D150A mutant retained PhoR interaction. We also tested these plasmids expressing the T25-PhoU fusions for interaction with a T18-PstB fusion protein. Interestingly, the A147 and R148 mutant proteins maintained interactions with PstB  that identifies covariance between residues. DCA identifies which residues from a sequence tend to co-evolve with any other residue by measuring the predictive power of one residue on another. This can identify direct interactions between residues, such as residues that are directly involved in protein/protein interactions. Also, residues that are involved in indirect interaction  and mechanistic interactions (as would be found in residues associated with an enzyme active site) are identified with DCA . Mutual phoU and phoR sequences from each species with 20 ala residues in between the two genes to aid in alignment. We aligned the sequences and then compared all the residues with DCA.We analyzed sequences of PhoU and PhoR from 196 gammaproteobacteria. We concatenated the Our previous work identified PhoU A147 and R148 residues as being involved in interaction with PhoR. To isolate residues from the PhoR PAS domain that show covariance with PhoU A147 and R148, we sorted the top co-varying residues of the PhoR PAS domain by MI for both A147 and R148 positions of PhoU. The top ten hits are shown in Table With the docking model it appears that the loops on the opposite side of PhoU from the PAS interacting surface are in close proximity to the CA domain of the opposite PhoR of the dimer. To observe any covariance between PhoU and PhoR at these surfaces, we compared the PhoU E121 residue to the CA domain with DCA. We see that the top PhoR CA domain residues (shown as green spheres) that co-vary with E121(shown as blue spheres) cluster at the surface of PhoR nearest to PhoU E121 Table .For a more complete analysis, we also sorted the potential interacting pairs by DI and found that several of the same residues were found in the top pairs Table . In the We noticed that there are few residues that appear to interact based on the docking model and DCA that did not show loss of function in our scanning mutagenesis (~PhoR163-166). To better understand these results we looked closer at the residues in our docking model structures. When we highlight the side chain of the PhoU R148 residue and the side chains of residues in positions 163, 164, and 166 of the PhoR PAS domain, it appears that the R148 may interact with the amino acid backbone for these sites and not the side chain residues as they appear to point away from the PhoU structure Figure . ChanginAdditionally, when distances between predicted interacting pairs is measured, several residues are found near R148 (within ~8Å) but few are found that near to A147. One explanation for this is that PhoU A147 itself does not directly interact with PhoR, but mutations of PhoU A147 may affect the ability of R148 to interact with PhoR. For example, the A147E mutation places a large acidic side chain right next to the R148 basic side chain and may disrupt proper PhoU/PhoR interaction and phosphate signaling. Using MI to sort our DCA results allows for identifying residues like this that are important for proper interaction but may not be directly involved. This phenomenon may also explain some of the PhoR PAS residues identified with DCA, like PhoR G118. The PhoR G118 residue is not found very near to PhoU R148 or A147 in our docking model Table . HoweverOur results confirm that PhoU is necessary for proper signaling in high phosphate, but that the PstSCAB transporter is also necessary. We have identified the A148 and R149 residues of PhoU as being important for interaction with PhoR. Using scanning mutagenesis, we identified residues of the PhoR PAS domain essential for interaction with PhoU. A docking model was identified and tested with DCA. The docking model shows PhoU interacting with both the PAS domain and the CA domain of PhoR, pointing to a potential mechanism for PhoU mediated switching of PhoR's kinase to phosphatase activity.The switch from kinase to phosphatase activity for histidine kinases is dependent upon the conformation between the CA and DHp domains ,34. The These studies have combined genetic, mutagenesis, computer modeling, and DCA analyses in studying the molecular interaction of PhoU and PhoR. It will be interesting to apply these techniques to identify residues involved in other signaling pathways and develop interaction models. For example, PhoU interaction with PstB appears to be weaker than PhoU interaction with PhoR based on our BACTH Figure  and 6B, The authors declare that they have no competing interests.SGG planned, performed experiments, prepared, and edited the manuscript. JM performed DCA, helped with analysis of data, and aided in preparing the manuscript. TD, TR, and ME helped in the scanning mutagenesis. PR helped with bioinformatic experimental planning and analysis. WRM designed experiments, conducted experiments, aided in data analysis, and aided in preparing and editing the manuscript.Table S1. Primers used in this study.Click here for file


PRDM9 is the sole hybrid sterility gene identified so far in vertebrates. PRDM9 gene encodes a protein with an immensely variable zinc-finger (ZF) domain that determines the site of meiotic recombination hotspots genome-wide. In this study, the terminal ZF domain of PRDM9 on bovine chromosome 1 and its paralog on chromosome 22 were characterized in 225 samples from five ruminant species . We found extraordinary variation in the number of PRDM9 zinc fingers (6 to 12). We sequenced PRDM9 ZF encoding region from 15 individuals (carrying the same ZF number in both copies) and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports from other vertebrates such as mice, humans, equids and chimpanzees. ZF-encoding regions of the PRDM7, a paralog of PRDM9 on bovine chromosome 22 and on unknown chromosomes in other studied species were found to contain 84 base repeat units as in PRDM9, but there were multiple disruptive mutations after the first repeat unit. The diversity of the ZFs suggests that PRDM9 may activate recombination hotspots that are largely unique to each ruminant species. Genetic recombination during meiosis governs genetic variability of a species by shuffling of alleles between genes linked on the same chromosome. This confers each species the ability to withstand the pressure of natural selection . StudiesPRDM9 has three functional domains, an N terminal KRAB domain (promotes protein-protein binding), a central PR/SET domain (histone methyl transferase activity) and a terminal zinc finger (ZF) domain of cysteine2 histidine2 type . BindingThe most fascinating feature of PRDM9 is the zinc finger (ZF) domain which has a minisatellite-like genomic structure and is encoded within a single exon. Each zinc finger (84 bp or 28 amino acids long) is repeated in tandem at both the DNA and protein levels with almost perfect homology [There is a strong variability in the number of encoded zinc fingers in PRDM9 not only across species but also within species. The variation in PRDM9 zinc fingers can thus alter the location of genomic recombination hotspots . The recSalient features of evolution of PRDM9 are that the zinc fingers are evolving under positive selection and concerted evolution across many species, particularly at positions involved DNA-binding . The resMus m. domesticus and Mus m.musculus hybrids has been attributed to allelic differences in PRDM9 [Intriguingly, PRDM9 has also been recognized as the only known mammalian speciation or hybrid sterility gene. Spermatogenic failure and resulting sterility of some in PRDM9 . PRDM9 iin PRDM9 , 25, 26.Ovis aries, Capra hircus) as well as large ruminants  and secondly, to explore the existence of PRDM7, a paralog of PRDM9 in these species.Because of its important role in recombination and apparently noteworthy role in speciation, PRDM9 has become the focus of intense study in the last few years. Evolutionary dynamics of PRDM9 can only be unraveled by analyzing its allelic diversity in different species. Till date, diversity of PRDM9 has only been characterized in humans , 27, chiBos taurus, Bos indicus, Bos taurus- Bos indicus hybrid), 10 from yak (Bos grunniens), 20 from mithun , 45 from sheep (Ovis aries) and 45 from goats (Capra hircus) were collected specifically for this study with the help of veterinary doctors after obtaining permission from the respective State Animal Husbandry departments , goat  and sheep  and from one state each for Yak  and Mithun . One hundred and five samples from cattle (artments . Prior iBos taurus PRDM9 sequence (KJ020105). Primer sequences used for amplification were: F- TGCTCTCTGGCCTTCTCCAGTCAGAA and R- GCTGCAGTAATTCTCCTGTGAC. PCR was carried out on an Veriti 96 well thermal cycler (Applied Biosystems) in 25 μL reaction mixture containing 2.5 μL of 10X Taq reaction buffer, 0.5 μl of 10mM dNTP mix (Fermentas), 0.5 μl of 10μM primer (each) and 0.25 units of Taq DNA polymerase (Sigma). The PCR reaction cycle was accomplished by denaturation for 5 min at 95°C; 30 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension step at 72°C for 30 sec, with a final extension at 72°C for 10 min. PCR products were electrophoresed alongside DNA molecular weight marker in 1.5% agarose gel and visualized by staining with ethidium bromide. The amplified fragments were gel purified using PureLink Quick Gel Extraction Kit (Invitrogen Canada Inc.) and directly sequenced or subcloned into pTZ57R/T vector (Thermo scientific). Recombinant clones were selected and plasmid DNA was extracted. Multiple clones (2–3) were sequenced using Sanger sequencing to obtain the DNA sequence of the amplified products.The zinc finger domain of PRDM9 paralog on chromosome 1 was amplified from genomic DNA of cattle, yak, mithun, sheep and goat. The primers for this study were designed manually using the http://asia.ensembl.org/Multi/Tools/Blast?db=core) to perform pair-wise alignment with their corresponding genomes. The first gene hit with the highest score and lowest E-value was used to assign the name and identity of the sequence. The beginning and end of all zinc finger coding sequences were marked in the sequences by referring to chromosome location viewer of the ENSEMBL database. Accordingly, sequences were trimmed to remove ambiguities while maintaining the reading frame using BioEdit 7.0 Sequence Alignment Editor [The sequences were subjected to EBI- BLAT   was carried out to determine whether these sequences were subjected to positive selection. These test ratios were determined using software tool Codeml from user friendly interface PAMLX  vs M2a (selection) and M7 beta vs M8 (beta & ω) using F3X4 codon matrix with fixed alpha. Further, in case if positive selection was confirmed, Bayes Empirical Bayes approach was used to identify the specific sites exhibiting positive selection by referring to posterior probability values in the codeml output .Bos indicus when aligned with Bos taurus genome assembly UMD3.1, indicated first hit to be to PRDM9 (ENSBTAG00000004538) located on chromosome 1 (98.35% identity) whereas only 2/3rd of the sequence could align perfectly with PRDM7 sequence present on chromosome 22 with 93.5% identity  using primers flanking the zinc finger domain region. The designed primers amplified genomic DNA unambiguously yielding homozygous or heterozygous ZF domain length variants of PRDM9 in the samples analyzed. An interesting observation was that a low molecular weight product of uniform size was constantly present across different samples of a species in addition to high molecular weight products of PRDM9 which varied within as well as between species. Intriguingly, all animals were homozygous for the low molecular weight product in all the species. Both high and low molecular weight products from representative homozygous samples of all the five ruminant species were sequence characterized after gel elution. The sequences of the high molecular weight product (around 1000 bp) from identity . When thBos grunniens (yak) samples corresponded to allele A of cattle PRDM9 which has 6 zinc fingers but in Bos frontalis (mithun), two alleles (A and B) with 6 and 7 zinc fingers were recorded. However, in all the Bos species , ZF domain of PRDM7 was of the same size. Highest diversity of PRDM9 was observed in goats where five different PRDM9 alleles were observed and were designated as C, D, E, F and G with 8, 9, 10, 11 and 12 zinc fingers respectively. Various PRDM9 zinc finger domain length variants observed in different species are summarized in Tables Bos species, intermediate in sheep and highest in goats.In cattle, agarose gels resolved four different sizes of the PRDM9 ZF domain and these were named as A, B, C and D alleles of PRDM9. Sequence analysis revealed that alleles A, B, C and D contain 6, 7, 8 and 9 zinc fingers respectively. The size of PRDM9 allele in all the We sequenced the final exon of PRDM9, which contains the ZF domains in 15 individuals . The size of these sequences ranged from 921 bp to 1326 bp for A to G alleles in the analyzed species. The sequences were submitted to GenBank and accessions numbers were obtained .in silico translation of PRDM7 sequence revealed presence of multiple stop codons. Interestingly, the location of the stop codons was more or less at the same sites in all the three Bos species (large ruminants). In small ruminants also, the location of these disruptive mutations was quite similar . Zinc finger repeats (84 nucleotide stretches) were observed in all the species but  similar .In all the species analyzed, the first finger (23 amino acid residues) was found to be highly conserved and was placed far apart from the minisatellite like domain structure which exhibited high variability. Ruminant PRDM9 sequences revealed presence of 43 different ZF domains characterized by amino acid variations at specific positions -9, -5, -2, -1, 2, 3 and 6 . In all Zinc finger array of some of the sequenced samples contained repeated ZF domains. Domain 6 was repeated in both A and B alleles of mithun, domain 19 was repeated in B and D alleles of cattle and domain 41 was common in all goat PRDM9 alleles . In partPositions -1, 3, and 6 in the ZF domain of PRDM9 have been reported to show strong signals of positive selection in a number of species , 18, 19.When Zinc finger domain coding sequences were tested for selection by determining LRT using Codeml, highest LRT values were found in goat (28.84 for M2a vs M1a and 36.34 for M8(beta & ω) vs M7 model, both at 99% level of significance) and lowest LRT values (5.56 and 6.03) were obtained in mithun. Comparative analysis of site specific models  yielded Homologous recombination during meiosis leads to reshuffling of both maternal and paternal alleles, thus contributing to genetic diversity . Three sMeisetz) gene are generated by alternative splicing and they too lack the zinc finger repeats and code for a protein with only the KRAB and the PR domains [In this study, we assessed the DNA sequence diversity in the zinc finger domain of PRDM9 on bovine chromosome 1 and its paralog, PRDM7 on chromosome 22 in five ruminant species. We found that although the sequenced region of PRDM7 contained 84 base repeat units characteristic of the PRDM family but there were multiple disruptive mutations after the first zinc finger. Horse PRDM7 has also been reported to contain early stop codons in the ZF domain . In prim domains  reported domains . Therefo domains . This rePan genus [−5 per generation). These observations support immense diversity of ZF domain of PRDM9 across diverse taxa.PRDM9 ZF domains in ruminants showed remarkable numerical and amino acid composition variation. The number of ZF repeats varied from 6 to 12 and the number of different ZF domains was 43. Our results extend the spectrum of high variation of PRDM9 alleles reported in other organisms  suggestian genus , 28. Recan genus . Similaran genus . Diversian genus . Instabian genus . Recent an genus  reportedet al. [In the different ZF domains, 9 among the 21 codons of the ZF unit showed nonsynonymous variability in our study. The most diverse codons were at positions -1, 2 and 3 of the ZF unit with nine, five and nine variant amino acids respectively. At position 6, there were three variant amino acids . Position -9 was the least diverse with two major variant amino acids (G and R) with G being specific for large ruminants and R being specific for small ruminants. Studies in multiple organisms have suggested that PRDM9 ZF domains evolve under strong positive selection , 16. In et al.  also repet al. . TherefoThe DNA-binding specificity of PRDM9 is determined by the residues at positions -1, 2, 3 and 6 of each ZF . Since wBos taurus) and yak (Bos grunniens) [PRDM9 is the first (and to date only) locus associated with hybrid sterility in mammals . An inteunniens) . Pure yaunniens) . Lou et unniens)  exploredThe present study characterized PRDM9 on bovine chromosome 1 and its paralog PRDM7 in five ruminant species. Remarkable numerical and amino acid composition variation was observed in zinc finger domain of PRDM9 since 7 alleles with varying number of repeats (6–12) and 43 different ZF domains could be identified. Ruminant zinc fingers were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports in other metazoans. PRDM7 in the studied species was found to contain multiple disruptive mutations, also reinforcing similar observations in humans and equids.S1 Fig(TIF)Click here for additional data file.


Intuitive understanding of hypothetical genes’s signal peptide, transmembrane helix (TH) domain and LipoP box of any pathogenic microorganisms is a prerequisite for the rational development of drugs and vaccine candidates. A protein harboring these sequences may serve as receptor and conveyer for various biostimulations generated during host-pathogen interaction. Identification and annotation of these proteins may help in designing of novel drug or developing new vaccine candidates to control CT infections.http://bmbpcu36.leeds.ac.uk/cgi bin/sig_pred), siglanlP4.0 , TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and LipoP box (http://www.cbs.dtu.dk/services/LipoP/).We have retrieved whole sequence of CT serovar D/UW-3/CX from NCBI database. To identify secreted proteins, TH domain and lipoP box, we have exploited the following online softwares: sigpred  have signal peptide as well as TH domain. Additionally, among five, four proteins  also have lipoP box. Harboring TH domain sequences suggests that they may have either membrane spanning, extracellular or intracellular domain which may help in host-pathogen interactions and/or signal transduction. It is also possible that these proteins may be secreted and thereby induces host immune responses but it will need further validation.We consider that CT149, CT605, CT623, CT823 and CT858 may be secreted outside the pathogen and should be explored further to understand the pathogenesis of CT.


To help conserve energy some bacteria inhibit nitrogenase activity upon exposure to ammonium. The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 can synthesize three functional nitrogenase isoenzymes: a molybdenum nitrogenase, a vanadium nitrogenase, and an iron nitrogenase. Previous studies showed that in some alphaproteobacteria, including R. palustris, molybdenum nitrogenase activity is inhibited by ADP-ribosylation when cells are exposed to ammonium. Some iron nitrogenases are also posttranslationally modified. However, the posttranslational modification of vanadium nitrogenase has not been reported. Here, we investigated the regulation of the alternative nitrogenases of R. palustris and determined that both its vanadium nitrogenase and its iron nitrogenase activities were inhibited and posttranslationally modified when cells are exposed to ammonium. Vanadium nitrogenase is not found in all strains of R. palustris, suggesting that it may have been acquired by horizontal gene transfer. Also, phylogenetic analyses of the three nitrogenases suggest that VnfH, the target of ADP-ribosylation, may be the product of a gene duplication of nifH, the molybdenum nitrogenase homolog.In microbes that fix nitrogen, nitrogenase catalyzes the conversion of N Cells were transferred to sealed 27 mL tubes containing an argon atmosphere and allowed to recover for 60 min before H2 was measured by gas chromatography as previously described  Protein Assay was used to measure total protein from NaOH lysed cells  ECL femto-substrate was used for visualization.Protein for visualization of posttranslational modification of dinitrogenase reductase was prepared from cells that had been grown under NF conditions as previously described  occurs when 50% of the subunits are modified, as NifH is found as a dimer and modification of one subunit sterically blocks modification of the second subunit  are most closely related to the three corresponding nitrogenase isozymes from A. vinelandii DJ . Slightly more distantly related strains of R. palustris all lack V-nitrogenase (Oda et al. R. palustris may have acquired its V nitrogenase genes by horizontal gene transfer. Regardless of the evolutionary origin of the structural genes for the alternative nitrogenases, all three nitrogenases in R. palustris CGA009 rely on several common components for production of metal cofactors and their insertion into the holoenzyme (Schuddekopf et al. R. palustris, the structural genes for both alternative nitrogenases are tightly regulated such that they are only expressed when the Mo-nitrogenase is ineffective at meeting the fixed nitrogen needs of cells (Oda et al. Our data indicate that the activities of the R. palustris VnfH and NifH proteins appear more related to each other than to any other dinitrogenase reductase protein analyzed. This suggests that VnfH in R. palustris is the product of a gene duplication event. A possible scenario for this gene duplication is as follows. R. palustris CGA009 expanded its nitrogen fixation abilities by horizontally acquiring the vnf gene cluster, which includes structural as well as some accessory genes, perhaps from a species similar to A. vinelandii (Bishop et al. R. palustris NifH and possibly be less efficiently modified by DraT2, even though it has an identical site of modification (Fig.vnf and nif gene clusters could result in duplication of nifH. The ability of VnfH to substitute for NifH has been demonstrated in A. vinelandii (Chatterjee et al. While VnfD, AnfD, and NifD from a wide variety of organisms fall into clades separated by the metal they use at the active site Fig., the dinR. palustris are subject to posttranslational modification by ADP-ribosylation. This suggests for maximum hydrogen production, the posttranslational regulation mechanism must be removed in strains of R. palustris engineered to express any of the three nitrogenases.Here, we have presented evidence that all three nitrogenase isozymes from


Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult.in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays.Here we use The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours  in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells.For the first time this data associates altered CDK5 substrate phosphorylation with oncogenesis in some but not all tumour types, implicating altered CDK5 activity in aspects of pathogenesis. These data identify a novel oncogenic mechanism where CDK5 activation induces CRMP2A phosphorylation in the nuclei of tumour cells.The online version of this article (doi:10.1186/s12885-015-1691-1) contains supplementary material, which is available to authorized users. CDK5R1 and CDK5R2) , 43in vilude tau .All of the above data suggests abnormal activation of CDK5 increases the risk of, or aggressiveness of, specific forms of cancer. However there are also reports that pharmacological (roscovitine) or siRNA inhibition of CDK5 enhances the proliferation of the breast cancer cell lines MCF-7 and MDA-MB321, while application of carboplatin, a chemotherapeutic used in the treatment of breast cancer, induces CDK5 activation . SimilarAlthough this indicates a complex relationship between CDK5 activity and growth of different cancer types, the general theme is that tight regulation of CDK5 activity is important for normal cell physiology and that localised or temporal gain (or loss) of function is associated with abnormal cell proliferation. This complex relationship makes it vital to develop the means to accurately assess CDK5 activity in tissue to clarify the potential contribution that this kinase plays in tumourigenesis and whether it presents any novel opportunities for intervention.The aims of our study were to identify high-confidence substrates as biomarkers of CDK5 activity in tissue and use these surrogate marker(s) of CDK5 activity to establish whether CDK5 activity was altered in human carcinoma.Peptides  containing 2 % (vol/vol) B27 serum replacement (Invitrogen), penicillin , streptomycin , and 1 % (vol/vol) L-glutamine (Sigma). HeLa and tumour cell lines were maintained in DMEM supplemented with 4.5 g/L glucose, 10 % (vol/vol) FCS, 1 % (vol/vol) penicillin (100 units/ml)/streptomycin (100 μg/ml) at 37 °C in 5 % CO2.Embryonic primary cortical neurons were isolated from Sprague–Dawley rats at day 18 gestation. Briefly, following dissection, cortex was digested in 0.25 % trypsin in Hank’s balanced salt solution at 37 °C for 20 min. Cells were manually dissociated by trituration using a fire-polished Pasteur pipette and plated onto 0.01 % poly-l-lysine-coated coverslips at a density of 2–5 × 10Plasmids were introduced into cells using Lipofectamine 2000 (Invitrogen) as per manufacturers instructions. Cells were incubated for 4 h at 37 °C before the transfection medium was removed and replaced with 5 ml growth medium. Cells were then incubated overnight at 37 °C, prior to lysis or fixation as below.Cells were lysed in ice-cold lysis buffer (1 % (v/v) Triton X-100, 50 mm Tris–HCl, pH 7.5, 0.27 M sucrose, 1 mM EDTA, 0.1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.1 % (vol/vol) β-mercaptoethanol, and Complete protease inhibitor tablet ). Following centrifugation to remove insoluble material, supernatants were collected, and protein concentrations determined using the Bradford method.Neurons were fixed in 4 % (w/v) paraformaldehyde in PBS for 10 min at 4 °C, permeabilised with 0.1 % (v/v) Triton X-100 in TBS for 3 min at room temperature, blocked with 1 % (w/v) BSA in TBS containing 0.005 % (v/v) Tween-20 for 1 h at room temperature, and incubated with primary antibodies diluted 1:50 in PBS containing 5 % (w/v) BSA for 1 h at room temperature. Secondary antibodies conjugated to Cy-3 fluorophores were diluted 1:250 in PBS containing 5 % (w/v) BSA and incubated with neurons for 1 h at room temperature. Neurons were counterstained with 0.5 ug/mL DAPI solution (Invitrogen). Image acquisition was performed on a Leica SP-5 laser scanning confocal imaging system using 63× objectives.Ethical approval was obtained by review through the Tissue Access Committee of Tayside Tissue Bank  and the studies follow the Guidelines of the Declaration of Helsinki for the use of human tissues for research. Sections of formalin-fixed, paraffin embedded tissue were cut at a thickness of 4 μm, collected onto Polysine-coated microscope slides  and dried overnight at 37 °C. Sections were dewaxed in Histoclear, rinsed in alcohol and endogenous peroxidase was quenched with 0.5 % hydrogen peroxide (100 volumes) in methanol at room temperature for 35 min. After washing in water, antigen retrieval was performed by boiling sections in 10 mM citrate buffer, pH 6.0 for 15 min in a microwave. After cooling, sections were rinsed in PBS and blocked with 5 % normal serum in PBS containing 5 % (v/v) avidin  for 30 min at room temperature. Sections were washed in PBS and incubated with primary antibody in 5 % normal serum in PBS containing 5 % (v/v) biotin at 4 °C overnight. After washing in PBS, sections were then incubated with biotinylated secondary antibody (1:250) (Vector Laboratories) for 30 min at room temperature, followed by streptavidin complexed with biotinylated peroxidase  at room temperature for 30 min. The peroxidase complexes were visualized using 0.25 mg/ml diaminobenzidine tetrahydrochloride (DAB) (Sigma) in PBS containing 5 mM imidazole (pH 7.0) and 0.075 % hydrogen peroxide for 10 min at room temperature. Cell nuclei were counterstained with haematoxylin (Sigma), dehydrated through graded alcohols, cleared in HistoClear and mounted in DPX. Images were taken using a Spot Insight QE digital camera or slides were digitally scanned (x40) using an Aperio ScanScope XT.6 cells) were harvested in 0.05 % (w/v) trypsin-EDTA and pelleted at 500× g for 5 min, washed 2x in PBS before subcellular fractionation which was preformed to the manufacturer’s specifications (Thermo Scientific- Cell fractionation Kit).Adherent cells (1–10 × 106 CPM/nmol) as previously described  ATP , the gels were dried and radiolabeled bands visualized by autoradiography. Quantification of nmoles of phosphate incorporated was obtained by excising the stained protein band from the gel and counting in scintillation fluid.SDS loading buffer was added to cell lysates and samples subjected to electrophoresis on 4-15 % polyacrylamide gels (Invitrogen) prior to transfer to nitrocellulose using the XCell II blot module (Invitrogen). Blots were blocked in 5 % (w/v) milk in TBST  Tween-20) and incubated overnight at 4 °C with the primary antibody diluted in 5 % (w/v) milk in TBST. Blots were washed in TBST and bound antibodies were detected using secondary antibodies linked to a fluorescent conjugate dye. Blots were visualized using a LICOR Odyssey® Infrared Imaging System .GST-tau (0.5 μM) was incubated with either p25/CDK5 or p35/CDK5 and MgATP for 5, 20 or 60 mins. Reactions were stopped by addition of 4× SDS-PAGE sample buffer prior to alkylation. GST-tau was isolated by SDS-PAGE, identified by coomassie staining and the destained protein band digested with 0.1 ml 2 g/ml trypsin in 50 mM TEAB overnight. Digests were extracted with 0.1 ml acetonitrile, supernatants dried, dissolved in 0.1 ml 5 % acetonitrile/0.25 % FA and 15 μl of sample from each time point separated on a 150 x 0.075 mm nanoC18 HPLC column prior to analysis on an Orbitrap-velos mass spectrometry system as described previously . LC-MS dNuclear lysates were isolated as described above, and aliquots alkylated prior to separation by SDS-PAGE and either coomassie staining or western blot (with phospho specific antibodies to CRMP2 to identify CRMP2A). The protein band equivalent to the molecular mass of CRMP2A was excised and the destained protein band digested with 20 μl 12.5 μg/ml trypsin  in 20 mM ammonium bicarbonate overnight at 30 °C. To each digest 20 μl of 100 % acetonitrile was added and incubated for 15 min then the supernatant removed. 30 μl of 5 % formic acid was then added to each gel piece and incubated for 15 min prior to the addition of an equal volume of 100 % acetonitrile . This extract was then removed and pooled with the original extract from the digest. A further 10 μl of 100 % acetonitrile was added to each and incubated for 10 min prior to pooling with the previous 2 extracts. The pooled extracts were then dried down, resuspended in 10 μl of 5 % formic acid then diluted to 1 % prior to injection. 15 μl of sample from each time point was separated on a PepMap RSLC C18, 2 μM column (75 μM × 50 cm nanoViper) (Thermo Scientific) connected to an Ultimate3000 RSLCnano System (Thermo Scientific) coupled to a LTQ Orbitrap Velos Pro (Thermo Scientific) via a EasySpray source Thermo Scientific). Orbitrap Velos Pro .RAW data files analysed with Proteome Discoverer (Ver. 1.4.1) using Mascot (Ver. 2.4.1) as the search engine against the IPI Human Database and sequence of CRMP2A.t-test was performed. A p value of <0.05 was considered significant and p values are expressed in relevant figures using asterisks where * represents <0.05, ** represents <0.001, and *** represents <0.0001.All statistical analysis was performed using Prism 6.0 software . Calculation of the mean was used to determine central tendency and standard error of the mean was calculated to quantify the precision of the mean. For comparison of substrate phosphorylation following transfection of p35/CDK5 and p25/CDK5 with untransfected control, statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test as comparisons between each group. For comparisons between squamous cell carcinoma and adenocarcinoma, a student’s https://www.oncomine.org/) and COSMIC (https://www.sanger.ac.uk/research/projects/cancergenome/) to search for evidence of differential expression or mutation of CDK5 and its two partners CDK5R1 (p35) and CDK5R2 (p39) in a range of cancer types. In the Oncomine database of gene expression profiles, CDK5 mRNA levels are lower in brain cancers compared to normal brain tissues and higher in a variety of other cancer types, notably breast, lung and lymphoma compared to the corresponding normal tissues. CDK5R1 shows a similar pattern in these cancers, whereas CDK5R2 is not commonly altered . This suggests that genetic alteration and/or gene expression changes in CDK5 or its cofactors are not a common cause of, or contributor to, oncogenesis. However this does not rule out disease-related post-translational alteration in CDK5 activity, and thus we decided to investigate CDK5 substrate measurements as a means to assess CDK5 activity in human tumours.As an initial assessment of the potential roles of CDK5 in human cancers, we interrogated Oncomine  residue immediately C-terminal (+1) to the phosphorylation site of its substrates, while the presence of a C-terminal Arg/Lys residue (+3 > +4 > +2) enhances phosphorylation of substrates even further . Meanwhitro Fig. . CDK5 istro Fig. . Indeed tro Fig. . This qutro Fig. . In thistro Fig. . Surpristro Fig. . This dain vitro at least  is accounted for by Ser235/Ser202/Thr205 phosphorylation. This does not rule out additional sites on tau are phosphorylated in longer incubations, or in vivo. However we focused subsequent studies on tau phosphorylation by CDK5 only at these three residues. We investigated several additional proposed CDK5 substrates  by each CDK5 complex Fig. . Three mtro Fig. , and thiHuman CRMP2 or CRMP4 was co-expressed with or without the CDK5 catalytic subunit and either p25 or p35 in HeLa cells  and tau (at Ser235 and Ser202/Thr205), and thus these substrates are potential biomarkers of aberrant CDK5 activity.Next we used immunofluorescence to investigate whether our phosphospecific antibodies could selectively detect phosphorylation of endogenous CRMP or tau in primary cells Fig. . We incuTau phosphorylation at Thr205 but not Ser202 is reduced by CDK inhibition Fig. . HoweverNext we investigated immunohistochemical staining of phospho-CRMP2 and phospho-tau after the primary neurons were embedded in paraffin to accurately mimic the fixation and processing of human clinical tissues used in diagnostic histopathology Fig. . The intin vitro and cell based studies suggest that of all of the potential substrates examined CRMP2 phosphorylation at Ser522 is most worthy for investigation as a surrogate marker for altered CDK5 activity in tumour tissue.Therefore our Previous work had suggested that altered expression/phosphorylation of specific CRMP isoforms was associated with lung and breast cancer –54. TherCDK5 itself is not differentially expressed in lung cancer subtypes , covering an additional eight human tumour types  with almost half 48.1 %) of patient cases scoring positive and half of those with a quick score >3 (Tables 8.1 % of The immunostaining of the nucleus of tumour cells with the anti-pCRMP2 Ser522 antibody is an unexpected result as there is very limited evidence that CRMP2 enters the nucleus of cells (most work has been done in neurons). As far as we are aware there is currently only one report proposing that phosphorylated CRMP2 is in the nucleus, with phospho-509/514 of CRMP2 being detected by immunofluorescence in the nucleus of breast cancer cells .To confirm the nuclear CRMP2 localisation using biochemical techniques we selected three human lung cancer cell lines representing different subtypes of NSCLC. The EBC-1 cell line is derived from human lung squamous cell carcinoma , the A54The most abundant form of CRMP2 protein (62 kDa) is found only in the cytoplasmic fraction for all cell lines Fig. . HoweverWe demonstrate that an antibody that selectively detects a validated CDK5 phosphorylation site on the substrate CRMP2 robustly stains NSCLC, B-cell lymphoma and to a lesser extent breast carcinoma. Furthermore we show for the first time that it is a specific splice variant of CRMP2 that localises to the nucleus of cancer cells. We propose that CDK5 regulation of CRMP2A could contribute to cancer initiation and progression, and this is supported by recent evidence implicating CDK5 activity in taxol-induced cancer metastasis . Phospho


KD values. Furthermore, molecular dynamics studies showed that GAB1 PH domain underwent large conformational changes upon ligand binding. Moreover, these hits inhibited the phosphorylation of GAB1 and demonstrated potent, tumor-specific cytotoxicity against MDA-MB-231 and T47D breast cancer cell lines. This effort represents the discovery of first-in-class GAB1 PH domain inhibitors with potential for targeted breast cancer therapy and provides novel insights into structure-based approaches to targeting this protein.The Grb2-associated binding protein 1 (GAB1) integrates signals from different signaling pathways and is over-expressed in many cancers, therefore representing a new therapeutic target. In the present study, we aim to target the pleckstrin homology (PH) domain of GAB1 for cancer treatment. Using homology models we derived, high-throughput virtual screening of five million compounds resulted in five hits which exhibited strong binding affinities to GAB1 PH domain. Our prediction of ligand binding affinities is also in agreement with the experimental  In this paper, we described the identification and evaluation of a set of first-in-class potent inhibitors targeting a new cancer target, Grb2-associated binder-1 (GAB1), which integrates signals from different signaling pathways, and is frequently over-expressed in cancer cells. To achieve our goals, we have employed intensive computational modeling to understand the structure of the GAB1 pleckstrin homology (PH) domain and screened five million compounds. Upon biological evaluation, we found that several inhibitors that induced large conformational changes of the target structure exhibited strong selective binding to GAB1 PH domain. Particularly, these inhibitors demonstrated potent and tumor-specific cytotoxicity in breast cancer cells. This represents a groundbreaking discovery in targeting GAB1 signaling which may be used for cancer therapy, especially for triple negative breast cancer patients. As is known, triple negative breast cancers are more aggressive with poor prognosis and difficult to treat clinically in silico platform for drug discovery, and demonstrates that targeting the PH domain of GAB1 provides a promising and novel therapeutic strategy for cancer treatment.Unfortunately, no three-dimensional (3D) structure is available to date for GAB1 PH domain or any PH domain in complex with drug-like small molecules. Challenges remain for accurate structural prediction due to its low sequence identity (<30%) to other PH domains with known structures 1, β2, β3, β6, β7 and α1  to guide secondary structure prediction of new PH domain . As no reliable PSSMs for β4 and β5 were generated due to low sequence similarity, we used PSIPRED server PH domains are unique due to their conserved secondary structures and 3D folds, all with seven β-sheets and a C-terminal helix. However, the pairwise sequence identities among different PH domains are usually below 30%, and the loop regions are hypervariable in length and amino acid sequence S1 Fig. shows the sequence logos derived from the collected 34 PH domains, in which the size of residue indicates the relative frequency of that residue at the corresponding position. As expected, we found that most conserved residues are in the hydrophobic cores of PH domains. The residues responsible for phosphoinositide binding are generally located at β1[7], β2[2], β2[5], β3[4], β3[+1] and β71,2 loop landmarked by the conserved K14 and W26, similar to myosin X  to DAPP1  . Large fluctuations of the Cα atoms were only observed in the β1,2, β2,3 and β5,6 loops . The quality of the lowest-energy model was assessed by QMEAN S3 Fig.). In addition, the DOPE per-residue profile demonstrated a significant decrease in the DOPE scores at the β2,3 loop, β4,5 loop, β5, β5,6 loop and β6 for the refined structure compared with the initial homology model .We constructed 1,000 homology models of GAB1 PH domain in complex with inositol-tetrakisphosphate (IP4) based on the X-ray crystal structures of four aforementioned templates. After loop refinement and molecular dynamics (MD) simulation, we selected one reliable model in which IP4 binds stably to GAB1 PH domain with a minor fluctuation of phosphates (RMSF<1.1 Å), shown in 1,2, β3,4 and β6,7 loops. The 2-hydroxyl group of IP4 oriented towards the β1,2 loop, and the 3,4,5-phosphates intensively interacted with the aforementioned basic residues in the β1, β2, β4 and β7. Particularly, K19 and R23 in the β1,2 loop formed hydrogen bonds with 5-P and 1-P, respectively P2 or PtdInsP2. Strikingly, the sequence motif NKKEFE in the β5,6 loop folded into an additional α-helix, as we termed α′. This additional α-helix also occurs in phospholipase Cδ PH domain P3 binding 1,2 loop formed intensive vdW interactions with the β7 and inositol scaffold (Region II) due to the smaller side chains of those hydrophobic residues around β6,7 loop compared to IRS1. All these specific structural features intrinsically offered possibility of designing selective inhibitors against GAB1 over other PH domains, as further discussed in the ligand-induced conformational changes section.As illustrated by ectively Fig. 3B.S5 Fig.). The residues responsible for PtdInsP3 binding were predicted to be K14, K27, R29, Y47, K49, R58 and R92, consistent to the mutagenesis studies S1 Table.To identify novel inhibitors of GAB1 PH domain, we performed structure-based virtual screening using our MD-refined structural model. Additionally, a protein-based pharmacophore filter was derived using GRID method to select those high-throughput virtual screening hits of which the docked poses matched the pharmacophores in silico identified hits, we performed three types of experimental assays to evaluate their bioactivities: direct binding to GAB1 PH domain, inhibition of Y627 phosphorylation of GAB1, and cytotoxicity IC50 in triple negative MDA-MB-231 and T47D human breast cancer cells. Our experiments revealed that 10 out of 20 hits demonstrated submicromolar to micromolar binding affinity (<50 µM) to GAB1 PH domain measured by surface plasmon resonance (SPR). Among them, GAB-001, GAB-004, GAB-007, GAB-016 and GAB-017 demonstrated promising bioactivity in the subsequent in vitro assays  , but not to IRS1 PH domain. In addition, it inhibited Y627 phosphorylation and killed breast cancer cells at IC50 = 23.7±2.7 µM (for T47D). GAB-004 achieved similar binding selectivity as GAB-001, but had a stronger inhibition of pY627 (81%) and a lower IC50 (19.4±2.9 µM). Interestingly GAB-007 demonstrated weak binding (KD = 42.3±8.9 µM) and mild pY627 inhibition (53%), but it showed high cytotoxicity in both MDA-MB-231 (IC50 = 4.6±0.7 µM) and T47D (IC50 = 20.1±4.3 µM) cell lines , and were 5-fold and 28-fold more selective to the GAB1 than AKT, respectively. Consistent to their high binding affinities, GAB-016 and GAB-017 also inhibited over 80% of Y627 phosphorylation.GAB-001 exhibited selective binding to GAB1 . In addition, some compounds that selectively bind AKT PH domain  did not effectively kill MDA-MB-231 or T47D breast cancer cell lines at 50 µM (All the aforementioned active inhibitors showed potent cytotoxicity to cancer cell lines (T47D and MDA-MB-231). More excitingly the cytotoxicity is specific to cancer cells as the inhibitors exhibit little inhibition in the non-cancer MCF-10A breast cell line Fig. 5. S8 Fig.). In addition, MD simulations showed that GAB-007, GAB-010 and GAB-016 could form stable binding to IRS1 PH domain , consistent to the SPR results in To further investigate the structural mechanisms of our inhibitors to interact with the GAB1 PH domain, we performed MD simulations of the protein-inhibitor complexes . The principle of this approach has been described elsewhere w(ξ) and protein-ligand separation PMF w(r). The details of mathematical calculations were available in S1 Method, and the w(ξ) and w(r) plots for eight protein-ligand complexes were available in S10 Fig. and S11 Fig. As indicated by 2 = 0.85). One may notice that these predictions encompassed two different PH domain targets (GAB1 and IRS1) and a variety of ligand chemotypes. The good correlation between experimental binding free energies and predicted free energies implied that the predicted inhibitor binding modes by MD simulations were accurate.To add another layer of validation of the binding modes predicted by MD simulations, we calculated the absolute binding free energies of our inhibitors to GAB1/IRS1 PH domain using an in-house potential of mean force (PMF) method, which aims to circumvent the insufficient sampling issue by introducing hypothetical intermediate states representing the association pathway of ligand from the unbound “bulk” regions to the ligand-binding “site”  for both GAB1 and IRS1 PH domains.We have generated eight reliable PH domain-inhibitor complex models from MD simulations (listed in Region I is comprised of the conserved K14GAB1/K21IRS1 (β1[7]), K27GAB1/K21IRS1 (β2[2]), Y47GAB1/Y46IRS1 (β3[4]) and F94GAB1/F93IRS1 (β7[3]) (Region I (RMSD>2 Å) for both GAB1 and IRS1, as illustrated by the RMSD analysis . This moiety could form cation-π and hydrophobic interactions with the surrounding K14GAB1/K21IRS1 and F94GAB1/F93IRS1, respectively . We had mentioned that β1[7], β2[2] and β3[4] were the PIP3-binding residues, thus Region I conformational changes were attributable to the activities of the inhibitors. Generally, the conformational changes of Region I residues in GAB1 were more substantial than IRS1, except F94GAB1 (IRS1 (IRS1 is likely to further open the pocket to accommodate larger moiety such as phenylisoxazole (GAB-010), as other IRS1 Region I residues were less flexible.The  F94GAB1 Fig. 7A.B1  and R92GAB1/E91IRS1 (β7[1]). Compared with Region I residues, more significant conformational changes were observed in the Region II residues in the GAB1 PH domain (RMSD>2.5 Å)  (blue plots in IRS1 and E91IRS1 significantly restrained the fluctuation of these two residues (Movie S1 available at http://imdlab.org/supporting/PLOSCompBio), while the electrostatic repelling between R58GAB1 and R92GAB1 made these two residues more flexible (Movie S3). These findings imply that the flexibility of Region II residues of PH domain may correlate the size of binding group.The Region III is located on the solvent-accessible side of the β7, especially I92GAB1 or H92IRS1 (β7[3]) (Movie S3 available at http://imdlab.org/supporting/PLOSCompBio). In contrast, this region in GAB1 PH domain did not exhibit significant conformational changes when binding any inhibitor . This could be due to the crosstalk between c-Met and α6β4 integrin pathway Our results also showed that the triple-negative breast cancer cell line, MDA-MB-231, was more resistant to GAB1 inhibitors than ER-positive breast cancer cell line, T47D Table 1.50 against T47D and MDA-MB-231 breast cancer cell lines than the non-tumorigenic MCF-10A cell line , as demonstrated by ell line Fig. 5. apo-structure of the PH domain protein could undergo large conformational changes in three regions to accommodate different inhibitors. The side-chain conformations of the residues in Region I determines the binding of either multiple electronegative groups  or an aromatic moiety conjugated with a group containing H-bond acceptors , as shown in Region II and Region III depend on several critical amino acids on β4 and β7 and the length of β1,2 loop, respectively. The selectivity of PH domain inhibitors may be designed based on our modeling of the protein structures. For instance, GAB-010 is highly selective to IRS1 but no binding to GAB1 or AKT1, largely due to the short β1,2 loop. The knowledge that GAB1 PH domain undergoes conformational change upon ligand binding provides novel insights of guiding the future structure-based drug design efforts, and of course more experimental validation will increase our understanding of GAB1 structure and functions.Although PH domains have been intensively studied as cancer target for drug discovery, to date there are no available protein structures in complex with any drug-like small molecules. As mentioned, this has significantly limited the structure-based drug discovery efforts. In the present work, we utilized several inhibitors to investigate the dynamics of GAB1 PH domain and evaluate their selectivity in potential cancer cell inhibition. Interestingly, we found that the A collection of five million drug and lead-like compounds which were curated from various sources . These figures were generated using the WebLogo server A total of 65 high-resolution crystal structures of PH domains were obtained from PDB, and we curated 34 non-redundant proteins. Their PDB IDs are listed in 1,2 is important for phosphoinositide binding, especially for Group 1 PH domain 1,2 loop of top ten initial models  were subjected to ligand-steered refinement using built-in function of MODELLER. We selected five models out of the 100 generated loop models based on the overall DOPE scores S3 Fig.The sequence of GAB1 PH domain was retrieved from UniProt database (accession number Q13480) GOLD 5.1 Movie S1), GAB1-GAB-017 (Movie S2) and IRS1-GAB-010 (Movie S3). Each trajectory contained 1,000 snapshots which were taken every 50 ps. The ligands and the critical residues were in sticks, whereas the backbones of PH domain proteins were in ribbons. Starting from the docking conformation, these MD trajectories vividly demonstrated the conformational changes of the PH domain proteins upon ligand binding. The movies were available at http://imdlab.org/supporting/PLOSCompBio.In order to evaluate the selectivity of our inhibitors, we optimized the also PH domain (IRS1 or GAB1) -inhibitor complex structures using MD simulations. The starting conformation for MD simulation is the binding mode which obtained the best score in molecular docking. The MD simulations were performed in triplicates for 50 ns using the parameters described in “3D structure prediction of GAB1 PH domain in complex with IP4” section. We also generated the trajectory of GAB1-GAB-001 complex (ξ) relative to the reference ligand or the protein-ligand distance (r) was sampled by umbrella sampling and weighted-histogram analysis method (WHAM). The full description of this method is available in S1 Method. The experimental binding free energies were derived from experimental KD (or Ki) using the equation The routine of PMF-based computation of protein-ligand absolute binding free energy has been previously described E. Coli. Expression and purification of the recombinant proteins were performed as previously described KD were injected at a high flow rate (30 µL/min). Dimethylsulfoxide (DMSO) concentrations in all samples and running buffer were 1% (v/v) or less. For the competitive binding assays and Ki determination, PtdInsP3-biotin labeled liposomes  and SA chips were used with increasing concentrations of the compound tested. We did triplicate SPR assays for each concentration.The DNA sequences of human GAB1 and IRS1 PH domain  were cloned into pGEX-4T1 inducible bacterial expression plasmid  transformed into BL21 (DE3) 2 atmosphere. MCF-10A cells were maintained in MEGM with other conditions same as the cancer cell lines. Cells were passaged using 0.25% trypsin and 0.02% EDTA. Cells were confirmed to be mycoplasma free by testing them with an ELISA kit . Our hit compounds were freshly prepared in DMSO at a stock concentration of 10 mM. For the evaluation of cellular proliferation, a standard 96-well micro-cytotoxicity assay was performed as described in reference Two human breast cancer cell lines and one normal breast cell line were used for this study: T47D ductal breast epithelial tumor cell line, MDA-MB-231 epithelial tumor cell line and MCF-10A non-tumorigenic epithelial cell line . T47D and MDA-MB-231 cells were maintained in bulk culture in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4.5 g/L glucose, 100 U/mL penicillin and 100 mg/mL streptomycin in a 5% CO312-IRS-1/Total IRS-1 Meso Scale Discovery plate as described by the manufacturer. The plate was read using a Sector Imager 2400A instrument . For the measurement of GAB1 phosphorylation, T47D cells were treated as for the phosphorylation of IRS1 evaluation. Cell lysates were run on a 7% SDS-PAGE and membrane were probed with specific anti-phospho-Tyr627 GAB1 . Each experiment was performed at least three times.For all biological assays, hit compounds were added at 20 µM concentration directly into the culture media of the cells for 4 hr following a 16 hr incubation of T47D cells without FBS. Cells were stimulated with HGF for 20 min at 50 ng/ml. Following this treatment, cells were lysed as previously published S1 FigSequence logos for PH domain. Position-specific logos for the secondary structure elements of PH domain (generated by Weblogo server). β4 and β5 are not included because of the high variation within these two β-sheets. The size of residue indicates the relative frequency of the residue at the corresponding position.(PDF)Click here for additional data file.S2 FigBackbone RMSD and per-residue backbone RMSF for GAB1-IP4 complex. The simulation lasted for 20 ns. The mass-weighted average RMSFs were calculated for each residue based on backbone atoms. Atomic RMSFs of four phosphates in IP4 were shown in the last table.(PDF)Click here for additional data file.S3 FigQuality of GAB1 PH domain model. This model was subjected to energy minimization prior to quality assessment. (A) QMEAN; (B) ProSA; (C); Ramachandran plot and summary generated by PROCHECK.(PDF)Click here for additional data file.S4 FigComparison of DOPE score profiles. IRS1 (PDB ID: 1QQG), GAB1 homology model and the lowest energy GAB1 model with MD simulation refinement were compared. The MD refinement model was subject to 1000 steps energy minimization with ff99SB force field to remove transient structural defects. The secondary structure is annotated at the bottom.(PDF)Click here for additional data file.S5 FigIsovolume of GAB1 PH domain generated by GRID. The area favoring H-bond acceptor  was displayed in magenta surface, whereas area favoring hydrophobic  probes was displayed in green surface. The protein backbone is illustrated with cyan lines, and the ligand (IP4) is in blue sticks. The crucial residues are labeled around isovolume surface.(PDF)Click here for additional data file.S6 FigSPR of GAB-001, GAB-016 binding to GAB1 PH domain.(PDF)Click here for additional data file.S7 FigInhibition of S312 phosphorylation of IRS1. The percentage of inhibition values are calculated from three separated experiments.(PDF)Click here for additional data file.S8 FigBackbone RMSD of GAB1PH/IRS1PH – inhibitor complex.(PDF)Click here for additional data file.S9 FigSchematic representation of frame of reference used to define the position and orientation restraints. To reduce the degrees of freedom, P1, P2, P3 and L1, L2, L3 were employed to represent the coordinates of the protein and the ligand, respectively, as described in (PDF)Click here for additional data file.S10 FigLigand conformational PMF as a function of mass-weighted RMSD (ξ). The ligand in the bulk is in black lines, whereas the ligand in the active site is in read lines. (A–E). PMFs derived from GAB1-inhibitor complexes. (F–H). PMFs derived from IRS1-inhibitor complexes.(PDF)Click here for additional data file.S11 FigSeparation PMF as a function of L1-P1 distance (r). (A–E). PMFs derived from GAB1-inhibitor complexes. (F–H). PMFs derived from IRS1-inhibitor complexes.(PDF)Click here for additional data file.S12 FigPharmacophore of the inhibitors. Green: hydrophobic or aromatic region. Blue: the projection of H-bond acceptor.(PDF)Click here for additional data file.S1 TableDocking scores for 20 htis. The hits which are consistently active in three assays are labeled with bold IDs.(PDF)Click here for additional data file.S2 TableThe crystal structures employed to generate PSSM.(PDF)Click here for additional data file.S3 Table1, β2, β3, β6, β7 and α1).PSSM profile for PH domain (β(PDF)Click here for additional data file.S1 MethodTechnical details for computational modeling.(PDF)Click here for additional data file.


Dictyostelium discoideum revealed that PD mutants have different effects and most importantly they explained the G2019S-related increased LRRK2 kinase activity. Furthermore, the structure of Dictyostelium Roco4 kinase in complex with the LRRK2 inhibitor H1152 showed that Roco4 and other Roco family proteins can be important for the optimization of the current, and identification of new, LRRK2 kinase inhibitors. In this review we highlight the recent progress in structural and biochemical characterization of Roco proteins and discuss its implication for the understanding of the complex regulatory mechanism of LRRK2.Human leucine rich repeat kinase 2 (LRRK2) belongs to the Roco family of proteins, which are characterized by the presence of a Ras-like G-domain (Roc), a C-terminal of Roc domain (COR), and a kinase domain. Mutations in LRRK2 have been found to be thus far the most frequent cause of late-onset Parkinson’s disease (PD). Several of the pathogenic mutations in LRRK2 result in decreased GTPase activity and enhanced kinase activity, suggesting a possible PD-related gain of abnormal function. Important progress in the structural understanding of LRRK2 has come from our work with related Roco proteins from lower organisms. Atomic structures of Roco proteins from prokaryotes revealed that Roco proteins belong to the GAD class of molecular switches (G proteins activated by nucleotide dependent dimerization). As in LRRK2, PD-analogous mutations in Roco proteins from bacteria decrease the GTPase reaction. Studies with Roco proteins from the model organism  Parkinson’s disease (PD) affects 1–2% of the population above the age of 65 and is the second most common neurodegenerative disease . PD causLRRK2 belongs to the Roco superfamily of proteins, which constitutes a novel multi-domain family of Ras-like G-proteins . LRRK2 cDictyostelium discoideum . Remarkably, the slime mold D. discoideum contains 11 Roco family members. Based on domain topology, Roco proteins can be divided into three separate groups , and Regulator of G-protein Signalling (RGS) domains. Although there is a high variation in these additional regulatory domains among the Roco proteins, as described below, previous studies have shown that the structure, function and regulation of the catalytic core is conserved.So far it has been a major challenge to isolate sufficient high-quality recombinant protein of full-length LRRK2 and/or domains thereof. Therefore, important structural understanding has come from work with related Roco proteins from bacteria and scoideum . Roco pretazoans . Howevere groups . MASL beThe kinase domain of LRRK2 has been extensively studied after its discovery. Kinases transfer the γ-phosphate of ATP to a target protein. Phosphorylation of proteins as a regulatory mechanism was discovered by Krebs and Fisher (1956), in their studies of glycogen phosphorylase. Nowadays, it is known that kinases are essential regulators of almost every signal transduction cascade. Kinases can be classified into three groups, the majority belongs to the group of serine/threonine kinases, a much smaller amount to the class of tyrosine kinases and only a few are classified as atypical kinases . Dictyostelium Roco4 in its active and inactive state, gave insight into the regulation mechanism of this group of kinases . The smaller N-terminal lobe is mostly composed of anti-parallel β sheets and contains the conserved αC-helix. The bigger C-terminal lobe mostly consists of α-helices and contains the activation loop with the conserved N-terminal DFG motif. The ATP binding site is formed by a cleft between those lobes and forms the catalytic site of the kinase together with the activation loop and αC-helix. The formation of a polar contact between Roco4 Lys 1055 from the β3-strand and Glu1078 from the αC-helix is essential for correct positioning of the αC-helix. The DFG motif is essential for catalysis: the Asp makes contact with all three ATP phosphates either directly or via coordination of a magnesium ion; the Phe makes hydrophobic contacts to the αC-helix and the HxD motif and is responsible for the correct positioning of the DFG motif. One can distinguish two conformations: a DFG-in (active) and a DFG-out (inactive) conformation. In the structure of active (phosphorylated) Roco4 kinase, the activation loop is visible and ordered. In contrast, the activation loop is not visible and is flexible in the structure of inactive (dephosphorylated) Roco4 kinase . Roco4 Arg1077 is conserved in almost all Roco proteins and corresponds to LRRK2 Gln1918. Consistent with the proposed mechanism: the Roco4 double mutant Gly1179Ser/Arg1077Ala and the homologous LRRK2 double mutant Gly2019Ser/Gln1918Ala, in which the new hydrogen bond is no longer possible, have again normal wild-type kinase activity . The COR domain consists of two parts: the highly conserved N-terminal part interacts with the Roc domain and the less conserved C-terminal part functions as dimerization device. It seems rather unlikely that the human RocCOR tandem has a different folding than that of the bacterial Roco protein. Importantly, an overlay of the human Roc and the bacterial RocCOR structure revealed major clashes of the highly conserved N-terminal part of the COR domain with the swapped Roc dimer [Figure 3, , GEFs reduce this affinity by many orders and thereby promote nucleotide release. This subsequently facilitates binding of GTP, which is present in about 30-fold excess over GDP in the cytosol of the cell . The intDicyostelium Roco1, additional stimulation of the already high intrinsic nucleotide exchange rate by GEFs might be required. It is well known that LRRK2 and other Roco proteins are active as a dimer  in the Roc domain and Tyr1699Cys in the COR domain . Due to  domains . Furtheractivity .In silico modeling predicts that this mutation changes the electrostatic surface of the domain . The COR domain functions as a dimerization device. Within the dimer, the Roco G-domains are flexible in the GDP-bound inactive state, but in the active form the G-domains come in a more fixed state in close proximity to each other. This conformational change is transmitted to other parts of the protein, which subsequently allows the activation loops of the two kinase protomers to be autophosphorylated and activated. The GTPase reaction is also critically dependent on dimerization, because efficient catalytic machinery is formed by complementation of the active site of one protomer with that of the other protomer. In this way the intramolecular GTPase reaction functions as a timing device for the activation and biological function of Roco proteins. The N- and C-terminal segments of LRRK2 regulate this intramolecular signaling cascade and are important for kinase activity, oligomerization, and/or localization. Most likely the N- and C-terminal protein-protein interaction domains are directly binding upstream proteins and/or downstream effector proteins and thereby determine the specificity of the Roco proteins.Altogether, the structural and biochemical data suggest that LRRK2 activity is regulated by at least two different mechanisms: intramolecular activation and binding of input/substrate to the N- and C- terminal domains (The multiple allosteric and enzymatic functions within one protein make LRRK2 an excellent therapeutic target. So far the major focus has been to develop kinase domain inhibitors as potential PD therapeutics. However, most of the specific LRRK2 inhibitors lead to kidney and lung abnormality. Furthermore, an increased kinase activity has only been thus far reported for G2019S. All other pathogenic mutations show inconsistent, modest or no effect on kinase activity. Importantly, the Roco structures show that PD-mutations have different defects in the LRRK2 activation mechanism. Therefore, alternative approaches that target other domains of LRRK2, including LRRK2, localization, dimerization, or allosteric modulation of the kinase domain may have significantly improved therapeutic benefits. To fully explore these potential targets more knowledge about the complex intramolecular activation mechanism of LRRK2, upstream and downstream regulators, and the cellular function of LRRK2 is needed. A high-resolution structural map of LRRK2 and related Roco proteins is essential in this enterprise.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.