Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a “basal body”, a lock-nut structure spanning both bacterial membranes, and a “needle” that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical  E. coli, Salmonella, Shigella, Pseudomonas aeruginosa, and Yersinia pestis are responsible for a wide range of diseases, from pneumonia to lethal diarrhea and plague. A common trait shared by these bacteria is their capacity to inject toxins directly inside the cells of infected individuals, thanks to a syringe-shaped “nano-machine” called the Type III Secretion System injectisome. These toxins lead to modifications of the host cell, allowing the bacteria to replicate efficiently and/or to evade the immune system, and are necessary to establish an infection. As a consequence, the injectisome is an important potential target for the development of novel therapeutics against bacterial infection. In this study, we focus on the basal body, an essential region of the injectisome that forms the continuous hollow channel across both membranes of the bacteria. We have used an array of biophysical methods to obtain an atomic model of the basal body. This model provides new insights as to how the basal body assembles at the surface of bacteria, and could be used for the design of novel antibiotics.Gram-negative bacteria such as  Salmonella enterica serovar Typhimurium SPI-1, Enteropathogenic Escherichia coli (EPEC) LEE and Shigella dysenteriae nomenclature, respectively) , is a specialized syringe-shaped protein-export system utilized by many pathogenic Gram-negative bacteria for the injection of virulence proteins (effectors) into host cells. Bacterial proteins destined for both needle assembly and host cell targeting are translocated via the injectisome in a process known as type III secretion ctively)  [4], [5]S. Typhimurium is an important medical pathogen causing gastroenteritis in infected individuals. Two of the major virulence determining factors are the discrete T3SSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2, which are required for bacterial invasion and replication within host cells Shigella dysenteriae T3SS S. Typhimurium basal body protein PrgH, in addition to the periplasmic domains of EPEC PrgK homologue EscJ and InvG homologue EscC Salmonella SPI-1 injectisome. Specifically, we report the crystal structures of the cytoplasmic domain of the IM ring protein PrgH11–120 , as well as the structure of the periplasmic domain of the OM ring InvG. , with the generalized symmetric modelling framework in the program Rosetta For both the cytoplasmic and periplasmic domains of PrgH, we applied a 24-mer oligomerization constraint, according to the established stoichiometry The stoichiometry of the T3SS OM secretin has been a matter of debate, with numbers between 12 and 14 proposed 11–120 structure was used in a similar conformation (data not shown), thus illustrating how the two-step modelling strategy allows for the discrimination between alternative ring arrangements. For molecular docking applications, the accuracy in the final docking solution is inherently limited by the precision in the backbone structure of the monomeric subunits. In the more general case an accuracy of 2 Å backbone RMSD or better in the coordinates of the monomer is needed to accurately predict the structure of the docked state To demonstrate the importance of using experimentally determined structures for our modelling protocol, we also generated ring models for the cytoplasmic domain of PrgH and the periplasmic domain of InvG using homology models based on the previously available structures MxiG Salmonella cultures. Mutants were designed using Rosetta to calculate maximal interface disruptiveness and by visual inspection of the models , is not required for assembly of the basal body  (PDB ID: 3GQS), Shigella (MxiG) Yersina (YscD) Shigella variant . FHA domains are frequently involved in interaction with phosphothreonine (pThr)-modified proteins. In Shigella, the PrgH orthologue MxiG was reported to interact with phosphorylated peptides from the secretion apparatus protein Spa33 Yersinia orthologue YscD revealed an unconserved phosphothreonine binding motif in vivo secretion assays  and EscJ do not form the conserved oligomerization interface and appear involved in intra-domain interactions or membrane association.We have previously observed the presence of a common modular domain, which we termed ring-building motif (RBM), in all the proteins comprising the basal body as well as components of the IM export apparatus 11–120, forms a hexamer in the asymmetric unit of the crystal . For the periplasmic PrgH170–392 domain, crystallographic symmetry-related molecules in the crystal produce a dimer that exploits the same general interface as the 24-mer biological assembly  plasmid (Novagen) fused to a 6xHis tag at the N-terminus followed by a thrombin cleavage site, using restriction-free PCR 11–120 was transformed into BL21(DE3) competent cells, and kanamycin-resistant colonies were used to inoculate LB media containing 50 µg/ml of kanamycin, at 37°C to an OD600 of ∼0.5. Expression was induced by IPTG at 0.1 mM, and the protein was expressed at 20°C for 20 hours. Cells were harvested, resuspended in buffer , lysed by sonication and debris pelleted at 45,000 g for 50 min. The protein was purified from the supernatant by passing it through Ni-activated chelating sepharose, and the His-tag was subsequently removed by adding thrombin (Roche) at 1∶1000 dilution for 16 hours at 4°C. The protein was further purified by size-exclusion chromatography using a Superdex75 gel filtration column . Expression and purification of PrgH170–392 was performed as described previously The gene coding for PrgH22–178 was amplified by PCR from S. Typhimurium genomic DNA and cloned into the pET-28(a) plasmid (Novagen) fused to a 6xHis tag at the N-terminus followed by a thrombin cleavage site, using restriction-free PCR22–178 was transformed into BL21(DE3) competent cells, and kanamycin-resistant colonies were used to inoculate LB media containing 50 µg/ml of kanamycin, at 37°C to an OD600 of ∼0.5. Expression was induced by IPTG at 1 mM, and the protein was expressed at 37°C for 5 hours. Cells were harvested, resuspended in buffer , lysed by sonication and debris pelleted at 45,000 g for 50 min. The protein was purified from the supernatant by passing it through Ni-activated chelating sepharose, and the His-tag was subsequently removed by adding thrombin (Roche) at 1∶1000 dilution for 16 hours at 4°C. The protein was further purified by size-exclusion chromatography using a Superdex75 gel filtration column .The gene coding for InvGFor SeMet-labelled protein, bacteria were grown in minimal media containing 100 mgs each of added L- Lysine, Phenylalanine, and Tyrosine; 50 mgs of L- Isoleucine, leucine, and valine; and 60 mgs selenomethionine per litre. Purification was performed as for unlabelled protein.11–120 (5–10 mg/ml) were grown at 20°C by sitting-drop vapour diffusion using 15–20% PEG 6000, 0.02 M CaCl2, 0.1 M HEPES pH 6.5 as reservoir solution. Crystals of PrgH170–392 were obtained at 20°C in a range of conditions using protein samples at 2 mg/ml concentration or less, and all requiring the presence of PEG precipitants. The crystals were all of a new crystal form . Crystals of PrgH22–178 crystallized in several conditions at 20°C, with pH above 7.5 and containing PEG precipitating agents, but formed clusters of needles not amenable for data collection. Single crystals could be obtained by decreasing the protein concentration to approximately 4 mg/ml, and the best diffraction was obtained for crystals grown in 100 mM HEPES pH 8.0, 30% jeffamine M-600. Selenomethionine-labelled protein crystallized similarly.InvG11–120, a mercury chloride derivative was obtained by soaking in cryo-protectant +2 mM mercury chloride for 10 minutes. A three wavelength MAD experiment was subsequently carried out as well as collection of a high-resolution native dataset in the absence of mercury chloride. For InvG22–178 a three wavelength MAD experiment on selenomethionine-derivative crystals was carried out, and a native dataset was collected.Crystals were cryo-protected by soaking in the crystallization condition supplemented with 30% glycerol and flash-cooled in liquid nitrogen. Data were collected at beamline 8.1 of the Advanced Light Source (ALS) and beamline CMCF-1 of the Canadian Light Source (CLS) at 100 K. For PrgH170–392, a molecular replacement solution was found with the program PHASER 11–120 and InvG22–178, structure determination and initial model building were carried out with the PHENIX suite 11–120 (FOM 0.51) and 6 Selenium sites for InvG22–178 (FOM 0.54). All models were further refined with REFMAC5 11–120, PrgH170–392 and InvG22–178 having respectively 96.46%, 98.1% and 97.9% of residues in the favoured region of the Ramachandran plot with no outliers. We note the presence of four PEG molecules and four phosphate ions per asymmetric unit in the PrgH170–392 crystal structure, for a total of 76 ion/ligand atoms. These molecules were present in the crystallization condition or cryoprotectant buffer, and are therefore not likely to be biologically relevant. The coordinates for PrgH11–120, PrgH170–392 and InvG22–178 have been deposited to the PDB, under the accession numbers 4G2S, 4G08 and 4G1I respectively.Data were processed and scaled with HKL2000 cytoplasmic, PrgHperiplasmic and InvG ring models have been deposited to the PDB, under the accession numbers 3J1W, 3J1X and 3J1V respectively.Rosetta modelling made use of standard symmetric docking protocols S. Typhimurium. Secretion assays were performed as described previously S. Typhimurium strains were grown at 37°C overnight, and cells were then pelleted at 6,000 g for 10 min. Proteins in the supernatant were precipitated by adding 10% TCA and pelleted at 6,000 g for 30 min. Pellets were washed in 0.5 ml acetone, re-suspended in 20 µl gel loading buffer, boiled, and ran in a 10% acrylamide SDS-PAGE gel that was stained with Coomassie Blue.For complementation assays, mutants were engineered into a plasmid containing the genes coding for PrgH or InvG, described previously S. Typhimurium lacking the gene for FliC and either InvG or PrgH Electro-competent strains of For EM analysis, purified injectisome samples were diluted in 10 mM Tris pH 8.0, 500 mM NaCl, 5 mM EDTA and 10 mM LDAO. They were prepared on carbon grids and stained with 0.75% uranyl formate using standard procedures. Images were collected with a H7600 Transmission Electron Microscope  equipped with a side mount AMT Advantage (1 mega-pixel) CCD camera (Hamamatsu ORCA), and operated at an acceleration voltage of 120 kV.−1 scan speed and a spectral bandwidth of 1 nm. Spectra were corrected for buffer.Circular dichroism (CD) spectra were recorded with a nitrogen-flushed Jasco J-810 spectro-polarimeter, at 20°C. Proteins were dialyzed against buffer containing 5 mM Tris pH 8 and 50 mM NaCl prior to analysis. 0.1–0.5 mg/ml protein was used with a path length of 0.1 cm. Data were recorded from 260 to 190 nm using a 2 s time constant, 10 nm minThe multiple sequence alignments  were made with ClustalW Figure S1Illustration of the 2-step ring structure modelling approach for the PrgH cytoplasmic domain and InvG periplasmic domain. The results of the fixed-backbone symmetric docking stage and for the perturbation docking calculation are shown for the PrgH cytoplasmic domain (A) and InvG periplasmic domain (B); RMSDs are computed for backbone atoms of the entire modelled 24mer complex for the fixed-backbone docking, while RMSD values are reported for a dimeric interface relative to the lowest-energy sampled model for the perturbation docking calculation. The EM map is used to restrain both steps of docking calculations, although in the last flexible-backbone stage the weights are reduced to one-half relative to the first, rigid-backbone step. (A) Two clusters of arrangements are identified in the fixed-backbone docking stage differing in the orientation of the C-terminus: in cluster 2, the orientation of the monomeric subunit is “flipped” relative to the center of the ring. Cluster 2 is a more collapsed ring that was excluded from further consideration on the basis of its poor fit to the EM map. Cluster 1 was then used as a seed for perturbation docking calculations in which the backbone degrees of freedom are also optimized to allow for more efficient energy discrimination. The final, low-energy ensemble  shows highly converged features in terms of the backbone conformation and side-chain packing along the interface. (B) Two clusters of arrangements are identified in the fixed-backbone docking stage differing in the orientation of the C-terminal domain: in cluster 2, the orientation of the monomeric subunit is “flipped” relative to the center of the ring. Cluster 2 was excluded from further consideration based on biochemical data (see the text). In the flexible-backbone step starting from cluster 1 , we obtain a highly converged low-energy interface .(TIF)Click here for additional data file.Figure S2The well-packed interface of the PrgH and InvG ring models. The ring models of InvG periplasmic domain , PrgH periplasmic domain  and PrgH cytoplasmic domain (C) are shown on the left, overlaid on the models reported previously  and (B) respectively). A close-up of the interface between molecules is shown on the right, with the interface side-chains apparent. For all three models, the interfaces are well packed, with a relevant network of interactions.(TIF)Click here for additional data file.Figure S3Mutants generated for the PrgH periplasmic domain. The location of all the mutated residues that were tested for secretion assay  is indicated on a PrgH molecule shown in cartoon representation, with the adjacent molecules in the ring model shown in surface representation. Only the G322Y mutant abrogated secretion (see tion see (TIF)Click here for additional data file.Figure S4Multiple alignment of the sequences from the secretins of several T3SSs. InvG_SPI1: S. Typhimurium SPI-1 secretin InvG; EivG_ETT2: Enterohaemorrhagic E. coli ETT2 secretin EivG; MxiD_Shi: Shigella secretin MxiD; YscC_Yer: Yersinia pestis secretin YscC; PscC_Pse: Pseudomonas aeruginosa secretin PscC; EscC_LEE: Enteropathogenic E. coli LEE secretin EscC; SsaC_SPI2: S. Typhimurium SPI-2 secretin SsaC. Strictly conserved residues are in a red box, similar residues are in red characters. The secondary structures for the periplasmic domains of InvG and EscC are in blue (top) and green (bottom) respectively.(TIF)Click here for additional data file.Figure S522–178, PrgH170–392 and PrgH11–120.Circular dichroism analysis of InvG Circular dichroism (CD) spectra of the crystallized domains of InvG and PrgH demonstrate the designed interface destabilizing mutants PrgH11–120 L87A (A), PrgH170–392 G322L (B) and InvG22–178 Q97L (C) have minimal effect on the overall secondary structure. CD is reported in mdeg and overlaid spectra have been corrected for protein concentration.(TIF)Click here for additional data file.Figure S6Fit of the ring models into the EM map. Close view of a monomer from the InvG (top), PrgHperiplasmic (middle) and PrgHcytoplasmic (bottom) ring models, into the EM map density (EMD-1875). Side views (left) and top views (right) are shown, and a lower contour level  compared to (TIF)Click here for additional data file.Figure S7Comparison with previous models. The RMSD between monomers in our PrgHperiplasmic and InvG models (green and orange respectively) and the models reported by Schraidt and Marlovits (grey) were calculated using PyMol. The difference in atom position is shown in yellow bars, illustrating the rotation in the PrgH model and the dramatic difference in the InvG model.(TIF)Click here for additional data file.Figure S8Comparison of the C-terminal domain or PrgH with its orthologues. Ribbon representation of PrgH11–120 (green), overlaid with the structures of its orthologues MxiG  from Shigella, and CT664/CdsD  from Chlamydia, viewed from the side of the beta-sandwich (left) and from the top (right). The common FHA fold is illustrated, with the variable single helix clearly apparent.(TIF)Click here for additional data file.Figure S9Multiple alignment of the sequences from PrgH orthologues in several T3SSs. PrgH_SPI1: S. Typhimurium SPI-1 protein PrgH; EprH_ETT2: Enterohaemorrhagic E. coli ETT2 protein EprH; MxiG_Shi: Shigella protein MxiG; YscD_Yer: Yersinia pestis protein YscD; PscD_Pse: Pseudomonas aeruginosa protein PscD; EscD_LEE: Enteropathogenic E.coli LEE protein EscD; SsaD_SPI2: S. Typhimurium SPI-2 protein SsaD. Strictly conserved residues are in a red box, similar residues are in red characters. The secondary structure of the cytoplasmic and periplasmic domains of PrgH is shown at the top (blue), and of the periplasmic domain of MxiG at the bottom (green). Red stars indicate proposed phThr-binding residues.(TIF)Click here for additional data file.Figure S10The putative pThr-binding residues of PrgH are not necessary for secretion. Coomassie-stained SDS-page gel of proteins secreted by S. Typhimurium strains with a chromosomal deletion of PrgH, complemented with a plasmid containing the PrgH gene or mutants. The flagellin protein FliC is used as a loading control. Secretion is not affected by the R35A, Q42A or D65A mutations, demonstrating that these residues are not essential for function.(TIF)Click here for additional data file.Figure S11Structural differences between InvG and EscC. Ribbon representation of the structure of InvG22–178 (orange), overlaid onto the structure of EscC21–174 (cyan). The overlay was based on the N1 domains, illustrating the shift in relative orientation for the N0 domain, viewed from the side (left) or front (right).(TIF)Click here for additional data file.Figure S1211–120 crystallizes as a hexamer.PrgH Ribbon representation of the PrgH11–120 crystal asymmetric unit. (A) The six molecules form a ring, with the helix located in the lumen. (B) Residue conservation is indicated on the surface representation, with the most conserved residues shown in red and the least conserved residues in blue. Conserved residues are primarily located at the interface between molecules, suggesting that this interface is biologically relevant.(TIF)Click here for additional data file.Figure S13Molecular basis for the charge repartition in the InvG ring model. Ribbon representation of the InvG22–178 monomer, with the residues forming the basic lumen of the InvG ring shown in blue, and those forming the acidic patch at the bottom of the InvG ring shown in red.(TIF)Click here for additional data file.Appendix S1Detailed protocol for the two-step modelling procedure. (A) Schematic representation of the procedure employed. (B) Command lines used in Rosetta for the modelling procedure.(DOC)Click here for additional data file.Video S1Ring models of the PrgH cytoplasmic domain (dark green), PrgH periplasmic domain (green) and InvG periplasmic domain (orange) docked in the EM map of the injectisome .(MOV)Click here for additional data file.


Enterobacteriaceae, there has been little appreciation for the distribution and diversity of these proteins amongst Gram-negative bacteria. Furthermore, there is little understanding of the molecular events governing secretion of these proteins to the extracellular milieu.Gram-negative bacteria have developed a limited repertoire of solutions for secreting proteins from the cytoplasmic compartment to the exterior of the cell. Amongst the spectrum of secreted proteins are the intimins and invasins  which are characterized by an N-terminal β-barrel domain and a C-terminal surface localized passenger domain. Despite the important role played by members of this family in diseases mediated by several species of the In silico approaches were used to analyze the domain organization and diversity of members of this secretion family. Proteins belonging to this family are predominantly associated with organisms from the γ-proteobacteria. Whilst proteins from the Chlamydia, γ-, β- and ε-proteobacteria possess β-barrel domains and passenger domains of various sizes, Int/Inv proteins from the α-proteobacteria, cyanobacteria and chlorobi possess only the predicted β-barrel domains. Phylogenetic analyses revealed that with few exceptions these proteins cluster according to organismal type, indicating that divergence occurred contemporaneously with speciation, and that horizontal transfer was limited. Clustering patterns of the β-barrel domains correlate well with those of the full-length proteins although the passenger domains do so with much less consistency. The modular subdomain design of the passenger domains suggests that subdomain duplication and deletion have occurred with high frequency over evolutionary time. However, all repeated subdomains are found in tandem, suggesting that subdomain shuffling occurred rarely if at all. Topological predictions for the β-barrel domains are presented.in silico analyses we present a model for the biogenesis of these proteins. This study is the first of its kind to describe this unusual family of bacterial adhesins.Based on our  Escherichia coli (Int) and Yersinia spp. (Inv). Intimins, first described by Jerse et al in enteropathogenic and E. coli strains, promote intimate bacterial attachment associated with attaching-effacing lesion formation in the gut mucosa Yersinia spp. to enter target cells, not by binding to a Tir-like protein, but by binding with high-affinity to multiple members of the β1-chain integrin family of mammalian cell receptors. The function of Invasin has been reiewed elsewhere.Pathogenic Gram-negative bacteria have developed many distinct secretion mechanisms for the efficient surface display of binding domains that specifically interact with their complementary receptors on host cell surfaces bacterial immunoglobulin-like (Big) domains decorated with a C-type lectin-like subdomain (CTLD).The Intimin and Invasin systems have been studied primarily with respect to their contribution to the virulence of Gram-negative pathogens. In contradistinction, little is known of the secretory mechanism of either Intimin or Invasin. These homologous proteins are related to each other both in terms of sequence and predicted structure, possessing a conserved modular organization The existence of a domain predicted to form a β-barrel pore has led to the suggestion that passenger domains may be secreted across the outer membrane in a manner similar to Autotransporter mechanisms. Like the Int/Inv family Autotransporters have a modular structure broadly consisting of an N-terminal signal peptide, a passenger domain and a b-barrel pore-forming domain. However, in contrast to the Int/Inv family, the signal peptide is juxtaposed to the passenger domain and the b-barrel is located at the extreme C-terminus. In the case of Autotransporters the b-barrel is proposed to mediate translocation of the passenger domain to the cell surface through the pore. Nevertheless, the evidence for an autotransporter like mechanism for the Int/Inv family of proteins is still equivocal being founded on analogy rather than empiric data Here we present evidence that the Int/Inv family is larger and more widely distributed among Gram-negative bacteria than previously appreciated. We demonstrate through phylogenetics that these systems have in general evolved in parallel with the organisms that utilize the systems and that there is only limited evidence for horizontal transfer. Finally, we probe the structures of the systems and propose a model for the biogenesis of Int/Inv secreted proteins.E. coli O157:H7  and the MakeTable5 program −5 cutoff and the GAP program with a 10 standard deviation (S.D.) cutoff −5 cut off to find homologous subdomains.In this study, intimin-γ of Topological predictions for individual proteins were made with the WHAT Chlamydiae, 69204798; 5) Chlorobi, 78186442; and 6) cyanobacteria, 78779562, 148241686 and 148243547. Redundant sequences and closely related sequences (of greater than 90% amino acid identity with a retained sequence) were eliminated, yielding 157 sequence-divergent proteins of sizes ranging from 237 amino acids  to 8620 amino acids. These 157 sequences were examined for the presence of the β-barrel domain. As the β-barrel is deemed essential for secretion and therefore a requisite feature of Int/Inv family members, all proteins lacking this domain were eliminated from further analyses. Sixty-nine sequence-divergent proteins resulted, all of which proved to have N-terminal  β-domains  only the Int/Inv proteins from γ-, β- and ε-proteobacteria as well as Chlamydia possess characteristic passenger domains with Big motifs, albeit these represent the majority of the proteins identified in this study; (iii) the β-barrel domains are conserved in size ranging from about 300–400 amino acids; (iv) in proteins lacking passenger domains, the functions of the β-barrel domains are unknown, but they may play roles in transporting other proteins such as non-covalent passenger domains (see below) across the outer membrane and/or anchoring them to the external surface of the envelope This series of investigations revealed several novel details including: (i) the Int/Inv family is larger than previously appreciated and is not limited to a few strains of http://www.biology.ucsd.edu/~msaier/supmat/IntInv) and phylogenetic trees  revealed that the phylogenetic relationships of the proteins within a cluster in general reflect the organismal phylogenies demonstrated by the 16S rRNA tree . The onlHaving established the phylogenies of the full-length proteins, we next examined the phylogenies of the β-barrel and passenger domains. We observed that the clustering patterns of the β-barrel domains are very similar to those of the full-length proteins. The only significant exception is cluster S, the members of which are found on two branches in the tree representing the β-barrel domain . This ise.g., within the last 1–2 billion years). The only possible exception is cluster G with one α-proteobacterial homologue and one β-proteobacterial homologue. These two proteins do not fall into either the α- or the β-proteobacterial cluster  and therefore may have been derived either by early gene duplication events or by lateral transfer from a dissimilar source.These data clearly suggest that between bacterial phyla and orders, there has been little or no lateral transfer of genetic material encoding members of the Int/Inv family of secreted proteins, at least over recent evolutionary time  for Sec-secreted proteins from Gram-negative bacteria. Some members of the autotransporter-1 , autotransporter-2  and the Two Partner Secretion  families possess extended signal peptides which adopt an unusual organization consisting of two charged domains, two hydrophobic domains and a signal peptide recognition site Pelodictyon luteolum. This hydrophilic domain (herein designated the α-domain) consists of one or two α-helical stretches predicted to extend from the outer membrane embedded α-domain into the periplasm.To probe the structural organization of the Int/Inv family of proteins, secondary structure predictions were made using the PsiPred program In the case of 19 proteins  include proteins with passenger domains of varying sizes. In these clusters, size variation among the members of each cluster is almost always substantial. We suggest that the latter proteins underwent slow evolutionary subdomain duplication/deletion/insertion compared to the former proteins.As noted previously, proteins in clusters E, G, H and J are primarily composed of β-barrel domains. While the remaining 48 proteins have C-terminal predicted passenger domains, the Pas1 and Cla1 proteins contain unique passenger domains, different from those in the other 46 passenger domain-containing proteins. The sizes of the proteins vary tremendously see . The homcids see . Branche−5 cut off), searching for subdomains common to at least two proteins or occurring at least twice within a single protein. Using this method, IntD0 and IntD1, and InvD1, InvD2 and InvD3 were found to be homologous repeats. Thus, for the purposes of this article, all of them are designated D0. The remaining IntD2, IntD3, InvD4 and InvD5 maintain the designations D2, D3, D4 and D5, respectively.The structures of the C-terminal passenger domains for an intimin and an invasin have been defined by Hamburger et al. (1999) and Luo et al. As a result of this approach, an additional 9 subdomains were identified and designated D6–D14. The positions and numbers of iterations of each subdomain is depicted in Tertiary structural predictions revealed that most of these subdomains adopt a structure similar to the Big subdomains . The pasTandem subdomain duplication and deletion is also likely to have occurred with high frequency for some subdomains  but not for others . These observations are not likely to have arisen by chance and therefore may have both physiological and mechanistic explanations. For example, the C-terminal subdomains in clusters A, N-R, and W are never duplicated. The lack of repetition most likely reflects the functional nature of the final subdomain. In contrast, many of the Big subdomains are repeated, an event which can be explained by the requirement of the Int/Inv protein to span the bacterial surface structures such that the functional domain can be displayed for interaction with the host. Interestingly, the final domain of an Intimin or an Invasin adopts a CLTD fold despite possessing limited amino acid sequence similarity.In all cases, a pair of disulphide-bonded residues are required to maintain function. In Intimin and Invasin, these have similar spacings . ExaminaYersinia frederiksenii in cluster A. Yfr4 proved to be in an operon with three other open reading frames (ORFs). While the invasin has 749 amino acids, the following three ORFs are of 301, 292, and 432 amino acids. TC Blast searches revealed that the first two of these proteins exhibited striking sequence similarity to the repeat units in the passenger domains of AT1 (1.B.12), AT2 (1.B.40), and the Int/Inv (1.B.54) families. The last ORF of 432 amino acids also contained repeats homologous to those described above, but these sequences were far more divergent than the first two ORFs. All four of the encoded proteins possess a signal sequence for export to the periplasm via the general secretory pathway as revealed by the use of SignalP . It seems reasonable that all four of these encoded proteins represent parts of the passenger domain associated with Yfr4. These observations provide the first evidence that a member of the Int/Inv family may function together with other polypeptide chains that serve to extend the passenger domain. All four of these gene products may play a role in Yersinia pathogenesis.Several of the proteins included in Aeromonas hydrophila in cluster T. The β-barrel domain protein, Ahy1, is encoded by the first gene of a four cistronic operon. The second and third genes both possess repeat subdomains typical of the Int/Inv family passenger domain. This therefore provides a second example where the two downstream genes probably provide the passenger domain function. Interestingly the fourth gene possesses the GGDEF domain and therefore is likely to be a diguanylate cyclase, which synthesizes cyclic di-GMP. This compound is believed to mediate the transition between planktonic growth and sessile biofilm formation . It is reasonable to suggest that the first three genes in this operon play an important role in biofilm formation, providing the function of intercellular adhesion. We therefore postulate that the operon is expressed under cyclic di-GMP control and is therefore silent in the planktonic state, but expressed during biofilm generation.A second example of this type proved to be Ahy1 from Erwinia tasmaniensis, also in cluster B, has a signal peptide as well as a passenger domain of moderate size.A very dissimilar example proved to be the ten short sequences included in cluster B. These proteins, which show homology with the β-domains of Int/Inv family members, range in size from 417 to 497 amino acids and lack a sizable passenger domain. They thus possess only a β-barrel domain. Surprisingly, we could not identify a signal sequence for these proteins using SignalP, suggesting that if they are exported to the periplasm they must use a pathway other than the general secretory pathway (Saier 2006). Indeed, these proteins lack the first 100 residues that include the signal sequences of other Int/Inv family members that are included in TCDB. This fact raises the possibility that these proteins are cytoplasmic. Genomic context for all ten of these proteins revealed that following the gene for the short putative Int/Inv family member (transcribed in the leftward direction) was a contiguous operon encoding a nitrate/nitrite sensor kinase/response regulator pair (transcribed in the rightward direction), followed by another continguous two cistronic operon encoding an nitrate/nitrite transporter and the α-subunit of a nitrate reductase (transcribed in the leftward direction). In this case we propose that the β-barrel domain serves a totally different function than in the other cases represented. Interestingly Eta2 from Salmonella enterica (cluster D) is also of a moderate size (660 amino acids). Consequently, we examined the genomic context of this protein as well. The Int/Inv family protein, Sen2, proved to be in a four cistron operon where the first gene encodes a putative lipoprotein, the second encodes the putative invasin, the third is a hypothetical protein of unknown function, and the fourth possesses a thioredoxin superfamily domain and has been annotated as a putative thiol peroxidase. The operon is preceded by a divergently transcribed araC-like gene encoding a transcription factor. The function of the invasin, while difficult to predict, might facilitate lipoprotein export, or serve as an anchor for it. Other short sequences listed in Sen2 of The analyses presented here reveal a conserved modular architecture for the Int/Inv family of proteins. Thus, members of this family possess (i) a signal sequence, (ii) a hydrophilic α-domain, sometimes decorated with a LysM domain, (iii) a β-domain with the propensity to adopt a transmembrane β-barrel conformation and (iv) a hydrophilic α′ domain. In addition, the majority of the Int/Inv family members possess a readily identifiable passenger domain that, based on analogy to well characterized Intimin and Invasin, is secreted to the cell surface. These observations allow us to propose a multi-step model for the biogenesis of Int/Inv proteins . (1) BasTopological predictions for the β-barrel domains suggest that these domains consist of 10 to 16 β-strands, with some being well-conserved and consistently predicted, while others are less certain. Structurally characterized transmembrane β-barrels are of 8 to 22 β-strands The β-barrel domains have been shown to form transmembrane ion channels and are thus predicted to function in passenger domain secretion across the outer membrane It remains unclear what the function of the α-helical α-domains is and what role the LysM domains play. The logical location for these domains is the periplasm. LysM domains are predicted to bind peptidoglycan and are found in peptidoglycan degrading enzymes The functions of passenger domain-less β-barrels are completely unknown. However, the β-domains undoubtedly provide one or more essential functions such as transport, anchoring, communication via ion channels and/or interaction with cytoskeletal elements underlying the cell membrane. Our genome context analyses suggest that they may be able to translocate/anchor non-covalently linked passenger domains to or on the cell surface as for Yfr4 and Ahy1. Other functions, for example, for cluster B homologues and Sen2 (see section entitled Genomic Context of Int/Inv Genes) seem probable. Perhaps the β-barrels can promote protein-protein interactions on both surfaces of the outer membrane. This possibility could introduce a means of direct communication from the exterior of a two-membrane cell to the periplasm or cytoplasm.The importance of size, sequence and domain variations in the dissimilar passenger regions of Int/Inv proteins has yet to be studied in detail. It is possible that the multi-subdomain passenger domains merely provide a rigid scaffold for the C-terminal adhesin, but this would not explain the varied compositions of these extracellular structures. Other possibilities include immune evasion for pathogens, escape from predators for free-living organisms, and contribution to their adhesive properties. Comparative analyses of the modular design of passenger subdomains, as recognized previously The studies reported here are the first of their kind to describe the unusual family of bacterial intimin and invasin adhesins. We hope it will provide a guide for future studies concerned with the structures, functions, mechanisms of action and evolutionary origins of these proteins.Figure S1Multiple alignment of the full-length Int/Inv family members.(5.07 MB PDF)Click here for additional data file.Figure S2Multiple alignment of N-terminal β-barrel domains.(0.37 MB PDF)Click here for additional data file.Figure S3Multiple alignment of N-terminal β-barrel domains. Predicted transmembrane β-strands are shaded yellow with predicted β-strands numbered 1 to 16 above the alignment. Fully conserved residues are colored red. Predicted α-helices are shaded blue located between β-strands 12 and 13.(0.14 MB PDF)Click here for additional data file.Figure S4Multiple alignment of C-terminal passenger domains.(2.82 MB PDF)Click here for additional data file.Figure S5Tertiary structural predictions of the Big subdomains comprising the C-terminal passenger domains.(0.78 MB PDF)Click here for additional data file.Figure S6Multiple alignment of passenger subdomain D0.(0.02 MB PDF)Click here for additional data file.Figure S7Multiple alignment of passenger subdomain D4.(0.01 MB PDF)Click here for additional data file.Figure S8Multiple alignment of passenger subdomain D5.(0.01 MB PDF)Click here for additional data file.Figure S9Multiple alignment of passenger subdomain D6.(0.01 MB PDF)Click here for additional data file.Figure S10Multiple alignment of passenger subdomain D7.(0.01 MB PDF)Click here for additional data file.Figure S11Multiple alignment of passenger subdomain D8.(0.02 MB PDF)Click here for additional data file.Figure S12Multiple alignment of passenger subdomain D9.(0.01 MB PDF)Click here for additional data file.Figure S13Multiple alignment of passenger subdomain D10.(0.01 MB PDF)Click here for additional data file.Figure S14Multiple alignment of passenger subdomain D12.(0.01 MB PDF)Click here for additional data file.Figure S15Multiple alignment of passenger subdomain D13.(0.01 MB PDF)Click here for additional data file.Figure S16Multiple alignment of passenger subdomain D14.(0.01 MB PDF)Click here for additional data file.Table S1The sixty-nine proteins of the Intimin/Invasin (Int/Inv) family included in this study, listed according to phylogenetic cluster and position within that cluster. Cluster designations refer to the clustering patterns in the phylogenetic tree shown in (0.12 MB DOC)Click here for additional data file.


C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ1-328-hFADS enzyme shows a slight, but not significant, change of Km values  and of kcat (4.2 × 10−2 s−1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ1-328-hFADS versus wild-type protein.FAD synthase  is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the  Two eSaccharomyces cerevisiae and named FMN1[FAD1[Candida glabrata[The first eukaryotic genes coding for monofunctional RFK and FADS were identified in amed FMN1 and FAD1FMN1[FAD1, respectFMN1[FAD1,7. Its sFMN1[FAD1, as had  glabrata. Thus, F glabrata,11.FLAD1, which is localized on chromosome 1 [N-terminal region of hFADS1. The first 17 residues  of hFADS1 represent a putative mitochondrial targeting peptide as predicted by bioinformatic analysis [Homology search using Fad1p as a template led to the identification of the human gene for FADS, named mosome 1 ,13. Two mosome 1 ,13. hFADanalysis . The mitanalysis .http://www.ensembl.org/index.html); for better detail, see [The human cytosolic FADS, hFADS2, was produced and purified to homogeneity as a recombinant His-tagged protein in . The enzail, see .N-terminus of the human enzyme, which resembles a molybdopterin-binding domain (InterPro database accession number: IPR012183). This domain is similar to Arabidopsis thaliana Cnx1G , Drosophila melanogaster cinnamon protein  and human gephyrin . This domain is also present in the Escherichia coli monofunctional mogA  and moaB  proteins, which are involved in molybdenum cofactor biosynthesis, the prosthetic group of molybdoenzymes, such as sulfite oxidase and xanthine oxidase , FADS differs from its yeast counterpart as being organized in two domains. Besides the PAPS reductase domain (InterPro database accession number: IPR002500), it contains an additional domain, localized at the ase see ,18). The. TheN-teper se. Furthermore, we investigated the possible role of the N-terminus resembling molybdopterin-binding domain on catalytic activity.The human PAPS reductase domain of hFADSs shares 34% identity and 60% similarity with the corresponding yeast Fad1p domain. Thus, here we studied whether, as for yeast, the PAPS reductase domain of the hFADS is sufficient to catalyze FAD synthesis, C. glabrata and Fad1p from S. cerevisiae. The alignment showed a consensus sequence of 64 amino acids (corresponding to 24% of the sequences lengths) among the 329–587 region of hFADS1 and the other two proteins (C. glabrata (PDB code: 3G5A) (MolProbity web service (MolProbity score: 2.98) [10-helices. The folding of the PAPS reductase domain resembles those of the related enzymes FMNAT of C. glabrata and Fad1p from S. cerevisiae (Cα atoms of the PAPS reductase domain (from Q70 to K232) on FMNAT of C. glabrata, calculated by Swiss-PdbViewer 4.0.1. . The structure has a prevailing α/β fold with central β-sheets surrounded by α-helices, and the region containing the active site seems to be conserved in the three enzyme structures as it appears from the comparison of C. glabrata and S. cerevisiae. In analogy to the FMNAT of C. glabrata[To identify which of the two domains of hFADS is responsible for FAD synthesis starting from FMN, a multi-alignment was performed among the hFADS1 , FMN adenylyltransferase from proteins . The mosproteins . On the e: 3G5A) . The mode: 2.98) . In thisrevisiae , in agre1-328-hFADS construct, encoding the PAPS reductase domain of the hFADS1 fused with the extra N-terminal sequence MSPIH6LVPRGSEASNS, was used for heterologous expression in the E. coli Rosetta (DE3) strain. The expression of the Δ1-328-hFADS protein was performed starting from conditions previously optimized for hFADS2 [d-galactoside (IPTG) at 20 °C. Electrophoresis of the IPTG-induced and un-induced cell lysates showed a heavily stained protein band mostly present (more than 70%) in the soluble fraction of the induced cell lysate (1-328-hFADS (31499 Da) calculated by the Expasy tool Compute pI/MW at the web site http://www.expasy.org. The soluble fraction, corresponding to lane 5 of The recombinant pH6EX3-Δr hFADS2 . Optimal1-328-hFADS was then identified with anti-FADS polyclonal antibody raised against recombinant hFADS2, produced according to [1-328-hFADS  of 0.24 ± 0.06 μM and 6.23 ± 1.5 μM for FMN and ATP, respectively, and with a kcat value of 0.041 s−1 , with 50% of maximum activity (Mg2+50) at 0.05 mM and maximal activity at 0.5–1.0 mM. The distinctive feature of the recombinant Δ1-328-hFADS was a decrease of synthesis rate at higher MgCl2 concentrations  was sensibly different from the entire hFADS2 (dashed line). In this case the sigmoidal shape of the Mg2+ dependence of the wild-type protein was transformed in a hyperbolic one with Mg2+50 of about 0.2 mM. Moreover, the rate of the reverse reaction, catalyzed by the recombinant Δ1-328-hFADS, decreased at MgCl2 concentrations above 1 mM. The reduction of reaction rate was even more evident than in the forward reaction with an activity at 5 mM MgCl2 of about half with respect to the maximal one, at 1 mM MgCl2.As in the case of the entire hFADS2 , the recn of FMN . The dep0032 s−1 . An averntration  catalyze2 or CaCl2 was studied in the absence of MgCl2 , but not for the wild-type enzyme (dashed lines).The dependence of the FAD synthesis reaction rate on the concentration of CoCl1-328-hFADS. While no significant changes were found in competitive inhibition by GDP (data not shown), the Dixon plot reported in  and the entire protein 6-His-hFADS2 [In s-hFADS2  is reporS. cerevisiae FAD1 gene as a template [C-terminus of isoform 1 and 2 of the human enzyme.The identification of human enzyme responsible for synthesis of FAD, coded by the gene FLAD1, was achieved some years ago by homology searching using the product of template . It is nS. cerevisiae and the FMNAT of C. glabrata are very similar to each other, the human PAPS reductase domain shows much less identity with the enzymes of the lower organisms. However, some of the important structural features of the enzymes are well conserved, also in the human one, such as the PP-loop and ARG1, which are involved in ATP binding, the flavin motif, that is involved in FMN binding and the γ-phosphate motif, whose role is still not clear [1-328-hFADS. Indeed, the tertiary structure and, hence, the folding of the enzyme may be better conserved than the primary structure as it appears from the comparison of homology structural model of the human PAPS reductase domain with the structure of the lower organism enzymes [It is not surprising that while the Fad1p of ot clear . The str enzymes . These o1-328-hFADS, corresponding to the PAPS reductase domain of hFADS, which was purified in a holo-form containing FAD tightly bound.Here, we report the characterization of a His-tagged recombinant polypeptide, namely Δper se, is able to catalyze the typical reactions of the FADS: FAD synthesis and its cleavage. The turnover number of Δ1-328-hFADS (kcat 4.2 × 10−2 s−1) is even lower than that measured for the entire enzyme, thus leaving open the hypothesis that FAD release may represent the rate-limiting step of the whole catalytic cycle of FAD synthesis process [2+, Co2+ and Ca2+ and also to purine nucleotide GTP are quite different from those of the entire human enzyme. These diversities indicate that the lack of the molybdopterin-binding domain results in regulatory differences and, hence, it should play some roles in the regulation of the enzyme hFADS.Experiments of functional characterization let us conclude that the PAPS reductase domain,  process . AccordiN-terminus molybdopterin-binding resembling domain somehow hid the binding of the ions and, hence, of the substrates at the PAPS active site. On the other hand, during the reverse reaction, the N-terminus domain could participate in modulating some Mg2+-dependent conformational changes, accompanying the pyrophosphorolytic disassembly of FAD cofactor, presumably binding this ion in a different site. The increase of the inhibitory effect by Mg2+ and Ca2+ at high concentrations may be the consequence of their interaction with negatively charged residues of the protein available in high number, as revealed by the calculation of the molecular surface of the protein, leading to protein instability. This phenomenon may not occur or be limited in the entire enzyme by complete or partial occlusion of these interaction sites by the presence of the molybdopterin-binding domain.The truncated form resulted in being more sensitive to divalent ions at low concentrations. We could postulate that the Ki between the entire and the truncated proteins, we postulate that the N-terminus domain resembling a molybdopterin-binding domain in the human FADSs may contribute to allocating GTP in a site different from the substrate sites. The ability of the N-terminus molydbopterin-binding domain to bind ions Mg2+ and purine nucleotides is not surprising, since the functional role of this domain in mogA and moaB proteins is to catalyse, in a Mg2+ and ATP-dependent way, the adenylation of molybdopterin [A. thaliana Cnx1 performs essentially the same function and is also responsible for copper coordination to molybdopterin-dithiolate sulphurs; its crystal structure has been solved in its apo-form (PDB: 1UUX) and in complex with molybdopterin and adenylated-molybdopterin (PDB: 1UUY) [Drosophila cinnamon gene, too, is functional homologous to the Arabidopsis Cnx1 [Based on the uncompetitive type of inhibition and on the different dopterin . The CnxB: 1UUY) . The Drosis Cnx1 .i.e., the covalent insertion of a molybdate ion into molybdopterin, with the molybdopterin binding domain located in the N-terminus part of the protein, performing an adenilyl transferase activity [In humans the multifunctional protein gephyrin is thought to catalyze the final step in molybdenum biosynthesis, activity . Howeveractivity  and for activity , thus leN-terminus domain, resembling a molybdopterin-binding domain, present in mammal FADSs, still remains to be investigated in further detail. We do not known whether and how the full-length hFADS protein is able to catalyse adenylation of molybdopterin, and at the moment, we have no idea about the structural relationship between this domain with the PAPS reductase domain in the hFADSs, but the ability to modulate GTP and divalent ions response introduces a novel role for molybdopterin-binding domain in the regulation of flavin cofactor biosynthesis.The function of the N-terminus portion of this domain generated by alternative splicing of the FLAD1 gene  differs in hFADS1 with respect to hFADS2, and it is surely involved in protein sub-cellular localization [Finally, we would like to point out that the lization . This aslization , is a ma1-328-hFADS may constitute a tool for high-throughput screening of toxicity of specific inhibitors/modulators that inactivate the microbial FAD-forming enzymes, but not the human ones.Since FAD-forming enzymes are strictly required for microbial viability ,16, and Escherichia coli Rosetta (DE3) strain was purchased from Novagen . Restriction endonucleases and other cloning reagents were purchased from Fermentas . Bacto Peptone and Bacto Yeast extract were from Difco . Chelating Sepharose Fast Flow was from Amersham Biosciences . The dye reagent for protein assay was from Bio-Rad .All chemicals were from Sigma-Aldrich , unless otherwise specified. The C. glabrata, Fad1p of S. cerevisiae and hFADS1 by using ClustalW2 . The consensus sequence represented 24% of the 329–587 region of hFADS1.The PAPS reductase domain of the hFADS1 (amino acid sequence 329–587) was identified by multi-alignment among FMN adenylyltransferase from C. glabrata (PDB: 3G5A_A) as template [The identified human PAPS reductase domain sequence was modeled by Modeller 9.10 software  using the FMN adenylyltransferase from template .EcoRI and XhoI sites, respectively. The amplified DNA was cloned in the EcoRI/XhoI sites of the pH6EX3 expression vector. The resulting recombinant plasmid, defined as pH6EX3-Δ1-328-hFADS, carried the extra N-terminal sequence MSPIHHHHHHLVPRGSEASNS.To clone the PAPS reductase domain of the hFADS1, the corresponding region was amplified using the forward and reverse primers 5′-CCGGAATTCATCAGAGGAAGAAGGACCCCT-3′ and 5′-GACCCTCGAGTCATGTGCGGGAGTT-3′, containing the 1-328-hFADS plasmid by calcium chloride treatment. Selection of transformed colonies was performed on LB-agar plates containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. E. coli Rosetta (DE3) cells carrying the recombinant plasmids were inoculated in 10 mL of LB medium  supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol, and cultured overnight at 37 °C with rotary shaking (≅200 rpm). A 5 mL-aliquot of the cell culture was transferred to 0.5 L of fresh LB medium supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol and grown at 37 °C to A600 equal to 0.6–0.7. Then, 0.5 mM IPTG was added to induce the expression of the recombinant protein, 6-His-Δ1-328-hFADS. Growth was continued overnight at 20 °C, bacteria were harvested by centrifugation at 3000 g for 10 min at 4 °C and the pellets stored at −20 °C. The bacterial pellet (about 3 g wet weight) was thawed on ice for 15 min and resuspended in 30 mL start buffer  supplemented with 0.2 mL of Protease Inhibitor Cocktail  and 0.5 mM PMSF. Cells were disrupted by mild sonication at 4 °C  using a Branson Sonifier 250. The soluble and the insoluble cell fractions were separated by centrifugation of the cell lysate at 20,000g for 30 min at 4 °C. The pellet (inclusion bodies and cell debris), containing the insoluble over-expressed proteins, was re-suspended in 15 mL start buffer, aliquoted and used for SDS-PAGE analysis and FADS activity assay. The supernatant, containing the soluble over-expressed 6-His-Δ1-328-hFADS, was used for SDS-PAGE analysis, FADS activity assay and further protein purification (see below).Rosetta(DE3) strain was transformed with the pH6EX3-ΔE. coli Rosetta (DE3) strain transformed with the pH6EX3-Δ1-328-hFADS plasmid, was applied onto a Chelating Sepharose Fast Flow column (3 mL packed resin), previously charged with 250 mM NiSO4 according to the producer’s protocol, and equilibrated with the start buffer. The column was first washed with 30 mL start buffer, then developed with a step gradient of 50 mM, 150 mM, 400 mM and 500 mM imidazole in the same buffer. At each step of the purification procedure, the FADS activity was measured (see below) and the purity of the 6-His-Δ1-328-hFADS was checked by SDS-PAGE. Prior to storing or further processing, fractions containing the purified recombinant protein were desalted by gel filtration on a PD10-column in 40 mM Hepes/Na, 5 mM β-mercaptoethanol, pH 7.4. These protein samples were stable for at least 30 days at 4 °C.A 30 mL-aliquot of the soluble cell fraction, obtained from 1-328-hFADS was estimated by absorbance spectra, which were recorded on an Ultrospec 3100pro spectrophotometer (Amersham Biosciences), essentially as in [280 readings. Because A280 for both free FAD and Δ1-328-hFADS-bound FAD is 1.7-fold A450, the A280, actually due to the apo-protein, may be calculated from the Equation:Protein concentration was measured with the method of Bradford, using BSA as a standard . In an aly as in . To this280 , as calculated from the protein sequence by using the Expasy ProtParam tool . Measurements made by either the spectrophotometric or the Bradford method differ by no more than 7%.The protein concentration was then estimated by using ɛThe FAD/protein monomer ratio  can be also estimated from the absorbance spectrum by considering:450 FAD/ɛ280 apo-enzyme ratio.where 0.3 is the ɛ450 = 12.2 mM−1 cm−1 for FMN and 11.3 mM−1 cm−1 for FAD. Under the experimental conditions used here, the FAD fluorescence constant (KFAD) was about 10-times lower than that of FMN (KFMN). Thus, the rate of FAD synthesis, expressed as μmol FMN · s−1 · μmol Δ1-328-hFADS−1, was calculated from the rate of fluorescence decrease, measured as the tangent to the initial part of the experimental curve by applying the following equation:The rate of FAD synthesis was measured by means of the differential fluorimetric properties of FAD with respect to FMN, essentially as in . FluoresF is the decrease in the value of fluorescence expressed in arbitrary units, ΔK = KFMN-KFAD is expressed as mM−1, V is the volume expressed in mL, t is time expressed in s and m is the protein amount in μmol. For activity measurements, the purified protein fraction (0.32 nmol) was incubated at 37 °C in 50 mM Tris/HCl, pH 7.5, containing 5 mM MgCl2, 2 μM FMN and 100 μM ATP, except where differently indicated in the legends to figures.where Δ2 and of the substrates 0.5 μM FAD and 1 mM NaPPi, except where differently indicated in the legends to figures. The rate of FAD cleavage was expressed as μmol FAD · s−1 · μmol Δ1-328-hFADS−1, and was calculated from the rate of fluorescence increase, measured as the tangent to the initial part of the experimental curve by applying essentially the equation described in Section 3.8, except that ΔF was in this case the fluorescence increase expressed in arbitrary units.The rate of FAD cleavage was measured by means of the differential fluorimetric properties of FAD with respect to FMN, essentially as in ,27. The Proteins were separated by SDS-PAGE (15% T polyacrylamide), according to Laemmli . QuantitIn immunoblotting experiments, SDS-PAGE separated proteins were electro-transferred onto a PVDF membrane using a trans-blot semidry electrophoretic transfer cell (Sigma-Aldrich). The immobilized proteins were incubated over-night with a 3000-fold dilution of a polyclonal antiserum against the recombinant hFADS, as previously described in .Data fitting was performed according to either the Michaelis–Menten equation:or allosteric kinetics equation:WhereTo fit the experimental data and to obtain estimates of the kinetic parameters, use was made of the GraFit software .C-terminus half of human isoform 1 and 2 of FADS, which corresponds to residues 329–587 of hFADS1 .We presented here some features of the PAPS reductase domain, identified by bioinformatics and localized in the By the homology modeling strategy, we describe the structure of this domain and identify the protein motifs responsible for catalysis.1-328-hFADS has been over-expressed in E. coli in its His-tagged form and purified as a FAD-binding protein performing an enzymatic catalytic activity, per se, not dissimilar from that of the entire enzyme.The polypeptide named Δ1-328-hFADS, were changed with respect to the wild-type enzyme, thus suggesting a regulatory role for the lacking molybdopterin-binding domain, localized at the N-terminus of hFADSs.Some regulatory features, such as cations sensitivity and inhibition constant for GTP of Δ


In the past few decades, a number of studies have reported that accessory foramina are located on the internal aspect of the mandible, indicating their potential importance for effective and successful clinical procedures. The aim of this study is to evaluate adult, infantile, and fetal human mandibles for occurrence of accessory foramina. To our knowledge, this is the first time that an attempt has been made to examine their particular co-location using a systematic approach and perspective. A total of 397 human mandibles, including 299 adult, 18 infantile, and 80 fetal, were investigated macroscopically for the frequency, position, and diameter of accessory foramina. In 96 % of investigated adult mandibles, at least one accessory foramen was found. Foramina were located either superior or inferior to the genial tubercle, as well as lateral to the tubercle. Bearing in mind their usual location, four different types of coexistence of foramina were distinguished. Accessory foramina were also present in similar locations in infantile and fetal mandibles. Accessory mandibular foramina are constant structures of human mandible. Their frequency, size, and location vary depending on the type of the foramen. Observations on children and fetal mandibles showed no significant differences in evaluation of accessory foramina, with the exception of lower occurrence in this group of subjects. In the mandible, accessory foramina refer to all mandible openings excluding the mandibular and mental foramina, and the alveolar sockets Sutton . NumerouIt was reported that accessory foramina are present more often on the internal surface of the mandible Chapnick  than on A vast majority of researchers found the accessory foramen lying in the midline, either superior or within the genial tubercle, and named it therefore the lingual foramen  mandibles, 18 mandibles of children, and 80 fetal (from 18 to 38 weeks) mandibles. The bones were taken from a collection of the Department of Anatomy, Poznań University of Medical Sciences in Poznań, and from a collection of the Institute of Anthropology, Adam Mickiewicz University of Poznań.The topography and frequency of accessory foramina on the internal surface of the mandibular body were investigated macroscopically. The diameter of the foramina was established using scaled wires (Ø 0.06–1.2 mm). To establish the exact location of the foramina, the height of the mandibular body was measured from the lowermost point of the symphysis lying in the midline to the uppermost point of the alveolar part of the mandible, lying between the alveolar juga of the central incisors.The distance between the inferior border of the mandible and the foramen, and their location with respect to the genial tubercle, were established. The horizontal location of the foramen in relation to the mandibular body height was expressed in %. In case of lateral foramina, the distance from the midline to the foramen, as well as the distance from the inferior border of the mandible to the foramen, were measured to establish the position.The average height of the mandibular bodies was 29 mm (from 16 to 42 mm). The total number of accessory foramina found on the internal surface of the bodies in all adult mandibles was 700. The number of foramina ranged from 0 (in 12 mandibles) to 7 foramina (in 2 mandibles). In 288 specimens (96 %), at least one accessory foramen was present on the internal surface of the mandibular body.Accessory foramen within or superior to the genial tubercleAccessory foramen inferior to the genial tubercleAccessory foramina lateral to the genial tubercle Fig. Fig. 1AccThere were four different types of foramina found on the internal surface of the mandibular bodies:In 276 mandibles (92 %), at least one accessory foramen located in the midline was observed .Accessory foramen located in the midline and superior to the genial tubercle was observed in 256 (86 %) mandibles Fig. , and itsAccessory foramen inferior to the genial tubercle was observed in 179 (60 %) mandibles Fig. . It was Accessory foramina lateral to the genial tubercle were located uni- or bilaterally. There were never more than two lateral foramina in one mandible. In 133 mandibles, the foramen was observed on the left side, and on the right in 132 mandibles  cases Fig. Fig. 5TypType Ib: one single foramen located in the midline, superior to or within the genial tubercle (52 cases)Type Ic: one single foramen located inferior to the genial tubercle, observed in 12 (4 %) of the mandibles.Type I was present in 130 mandibles. It was subdivided into 3 groups:Type IIa was observed in 93 (31 %) mandibles. Two midline foramina were present , and they were accompanied by either one or two lateral foramina Fig. .Fig. 6TyType IIb, observed in 45 cases (15 %): midline foramen located superior to the genial tubercle, accompanied by either one or both lateral foraminaType IIc, observed in 8 cases (3 %): midline foramen located inferiorly to the genial tubercle, accompanied by one or two lateral foramina.Type II was characterized by the presence of accessory midline foramina accompanied by lateral foramina. It was observed in 146 cases. It was subdivided into 3 groups:In type III, only foramina lateral to the genial tubercle were observed. It was found in 10 mandibles (3.4 %). In 5 specimens, a single, unilateral foramen was present, whereas in 5 mandibles, bilateral foramina were found Fig. .Fig. 7TyType IV included 13 mandibles with no foramina.In the mandibles of 18 children that we examined, a total of 33 accessory foramina were found , at least one accessory foramen was observed, and in all cases, it was located in the midline, just above the genial tubercle Fig. .Fig. 9InAccessory foramen lying inferior to the genial tubercle was observed in 9 mandibles (50 %). In two cases, it was twofold. In 5 mandibles (28 %), foramina lying laterally to the genial tubercle were found, and they were bilateral in all cases. Type Ia was observed six times, and therefore with the highest frequency; type Ib and IIa were observed three times each, and type IIb was found twice. Types Ic, IIc, and III were absent. Type IV was found in 4 mandibles.Of the 80 fetal mandibles investigated—from 4 to 9 months intrauterine period—a total of 72 accessory foramina were found on the internal surface of the mandible Fig. . The numThe diameter of the foramina in fetal mandibles ranged from 0.06 to 0.3 mm.The accessory foramina in fetal mandibles formed type Ib in 14 cases, and type Ic and type III in 10 cases each. Type IIb was observed in 7, type IIc in 4, and type Ia in 3 mandibles. Type IIa was found only once. Type IV was found in 31 mandibles.In some fetal mandibles, it was observed that the accessory foramina opened into the short interosseous canal that was directed anterolaterally and joined the mandibular canal Fig. .Fig. 11FThe presence of accessory foramina has been reported in many descriptive reports. Numerous studies which examined foramina have confirmed their presence and, furthermore, discussed their potential role in neurovascular transmission and possible clinical significance (Longoni et al. A vast majority of the morphological reports confirmed that accessory foramina occur in nearly all investigated mandibles (Shiller and Wiswell It is reported that, in the majority of investigated mandibles, at least one accessory foramen located on the internal surface is present (Baldissera and Silveira Our results confirm the observations made (Baldissera and Silveira Taking all the accessory foramina into consideration, it seems that foramina located in the midline appear with the highest incidence rate. Accessory foramen located superior to the genial tubercle is the most frequent Eriguchi ; howeverOn the internal surface of the mandible, accessory foramina located lateral to the genial tubercle are observed with frequency ranging from 30 % (Fanibunda and Matthews Current literature shows no evidence of attempts to distinguish the different types of coexistence of accessory foramina. We observed that they form repeated patterns, which we referred to as “types” according to their location on the internal surface of the mandible.According to studies of the mandibular body, accessory foramina located in the alveolar part, between the alveolar sockets in the lower incisors region, were also distinguished. Detailed information, including the frequency, size, and topography of these foramina, was previously reported (Przystańska and Bruska The idea of conducting studies on the development process of the mandibles was inspired by the fact that accessory foramina are present in adult mandibles. The first, and thus far only, research which mentions accessory foramina in fetuses was a study by Chavez-Lomeli et al.  on the dIn conclusion, it can be stated that macroscopic observations revealed the presence of accessory foramina in most of the investigated mandibles, and thus the foramina can be considered as constant anatomical structures and their nomenclature should be unified and standardized.Ethical approval was obtained for the Department of Anatomy from the Institutional Review Board at Poznan University of Medical Sciences


Some components of the KKS have been identified in the fishes, but systematic analyses have not been performed, thus this study aims to define the KKS components in teleosts and pave a way for future physiological and evolutionary studies. Through a combination of genomics, molecular, and biochemical methods, we showed that the entire plasma KKS cascade is absent in teleosts. Instead of two KNGs as found in mammals, a single molecular weight KNG was found in various teleosts, which is homologous to the mammalian LMW KNG. Results of molecular phylogenetic and synteny analyses indicated that the all current teleost genomes lack KLKB1, and its unique protein structure, four apple domains and one trypsin domain, could not be identified in any genome or nucleotide databases. We identified some KLK-like proteins in teleost genomes by synteny and conserved domain analyses, which could be the orthologs of tetrapod KLKs. A radioimmunoassay system was established to measure the teleost BK and we found that [Arg0]-BK is the major circulating form instead of BK, which supports that the teleost KKS is similar to the mammalian tissue KKS. Coincidently, coelacanths are the earliest vertebrate that possess both HMW KNG and KLKB1, which implies that the plasma KKS could have evolved in the early lobe-finned fish and descended to the tetrapod lineage. The co-evolution of HMW KNG and KLKB1 in lobe-finned fish and early tetrapods may mark the emergence of the plasma KKS and a contact activation system in blood coagulation, while teleosts may have retained a single KKS cascade. The kallikrein-kinin system (KKS) consists of two major cascades in mammals: “plasma KKS” consisting of high molecular-weight (HMW) kininogen (KNG), plasma kallikrein (KLKB1), and bradykinin (BK); and “tissue KKS” consisting of low molecular-weight (LMW) KNG, tissue kallikreins (KLKs), and [Lys Briefly, 5 mg thyroglobulin was mixed with 1 mg [Arg0]-BK in 4 mL PBS (pH 7.0) and glutaraldehyde was added to a final concentration of 0.05 %. The mixture was incubated at 25 °C for 1 h with gentle agitation and reaction was stopped by addition of 0.1 mL ethanolamine. The mixture was dialyzed against 1 L PBS overnight at 4 °C and protein concentration was determined. For each injection, 0.2 mg conjugated protein was used and three rabbits were first immunized with the conjugated protein in Freund’s complete adjuvant followed by two booster immunogen injections in Freund’s incomplete adjuvant. The interval between injections was 4 weeks and the rabbits were sacrificed for final bleeds 3 weeks after the third immunization. [Arg0]-BK in blocking buffer for 16 h at 4 °C. To displace the bradykinin specific epitope, [Arg0]-BK were added to the primary incubation at 10-5 M final concentration. After the primary incubation, the membrane was washed three times with PBS-T and incubated with a secondary antibody 1:5000 Anti-Rabbit IgG, horseradish peroxidase-linked  in PBS-T. Chemiluminescent signal was produced by Western Lightning ECL reagent  and images were captured on X-ray films , which were developed in an in-house darkroom.  Total protein concentrations of the plasma samples from various fish species were determined by a BCA Protein Assay kit . Plasma samples (30 μg protein) were heat denatured under reducing conditions and resolved on a pre-casted 5-20% gradient SDS-PAGE  with constant voltage 150 V for 90 min. Resolved proteins were transferred to a 0.45 μm PVDF membrane  by a semi-dry western blotting system. The membrane was blocked by 4% skimmed milk in PBS-T  for 1 h at 25 °C and was subsequently incubated with 1:500 primary antiserum for -bradykinin (YRRPPGWSPLR) was radiolabeled by 125I using the lactoperoxidase method and the iodinated peptide was purified by an HPLC system. The 125I-BK was diluted in RIA buffer containing 0.1% RIA-grade bovine serum albumin to produce ~10,000 cpm per assay tube. The titers of the antiserum were determined by serial dilution and maximum binding was adjusted at 20 % of the total binding. Incubation of standard [Arg0]-BK or unknown samples, radiolabeled tracer, and antiserum  was carried out at 4 °C for 24 h. Separation of free and bound fractions was achieved by precipitation using a secondary antibody, goat anti-rabbit IgG , and 16% polyethylene glycol followed by centrifugation at 3,000 g for 1 h at 25 °C and then 30 min at 4 °C. Unbound fractions were removed by aspiration and specific binding in the pellet was measured using a gamma counter . A standard curve that ranged from 3 to 30,000 fmol/mL was constructed. All measurements were performed in duplicate. Synthetic -BK and BK were collected at 1 min intervals and dried by lyophilization. The retention times for [Arg0]-BK and BK in the HPLC system were confirmed by the injection of synthetic peptides. The lyophilized fractions were reconstituted in RIA buffer for assay. The plasma samples were purified by Sep-Pak C elution . The par0]-BK in plasma in vitro, 3 blood samples with inhibitor cocktail were prepared. Plasma fractions were obtained and divided into 0.2 mL aliquots. Synthetic [Arg0]-BK was added to the plasma aliquots to a final concentration of 3,000 fmol/mL and incubated at 25 °C for 0, 10, and 30 min. Incubation was stopped by the addition of equilibration buffer used for Sep-Pak cartridge purification. The immunoreactive (ir)-[Arg0]-BK was measured using the newly established RIA. In an additional validation experiment, the pooled plasma samples were spiked by synthetic [Arg0]-BK to final concentrations of 300, 1000, 3000 fmol/mL and then extracted as previously stated. The concentration of [Arg0]-BK in the HPLC fractions were measured to determine the percentage recovery. Synthetic -BK (BK) and -bradykinin were serially diluted in RIA buffer and measured by RIA to determine the cross-reactivities among these closely related peptides.To test the degradation rate of [Arg0]-BK during primary incubation, which demonstrated the specificity  of HMW KNG. The KNG in lamprey consists of one cystatin-like (cathelicidin) domain and a bradykinin domain, which is the simplest KNG structure in vertebrate so far [kng1 in teleosts possess two cystatin domains and a bradykinin domain. Coelacanths KNG possess two cystatin domain, a bradykinin domain, a histidine-rich domain, and a putative apple-interacting domain, which highly resembles the structure of HMW kng1 in mammals except the absence of D2 ( The domain structures of vertebrate e so far . From coce of D2 . Comparice of D2 . kng1 was cloned and sequenced (GenBank accession number: KF111560), and its mRNA distribution in various tissues was shown in kng1 was expressed in all tissues examined and the highest expression was found in liver. Three putative N-glycosylation sites (amino acid residue NXT or NXS provided that X is not a Pro) and 19 putative O-glycosylation sites that cluster at the C-terminal region were identified [2, Gly4, Pro7, and Arg9.  The eel entified . The molklkb1 is apparently absent in teleost lineage. In the phylogenetic tree  and the duplication was specific to the mammalian lineage. Three-dimensional protein modeling of KLKB1 of coelacanths showed the same structural arrangement to that of human: four apple domains followed by a trypsin domain. In teleosts, however, such protein architecture does not exist and the most closely related proteins are some skin mucus lectins, which consist of only four apple domains and are without a trypsin domain. Using human KLKB1 as a base, percentage identity matrixes of some representative species are shown (in green brackets) to indicate the general protein homology. In the synteny analysis  to indicate the general protein homology. Alignment of annotated KLK13 of several teleost species with human KLK1 showed that telelost KLK13 lacked the catalytic triads of the trypsin domain  than that employed for the RIA experiments . The western blot detected a single immunoreactive band at 54 - 60 kDa in each species, a result that demonstrates the lack of HMW KNG in teleosts at the protein level. The percentage glycosylation of KNGs in teleosts was similar to that of LMW KNGs in mammal . Histidine-rich domains (D5) in human and coelacanths are underlined.(TIF)Click here for additional data file.Table S1Primer sequences for PCR used in tissue expression profiling.(TIF)Click here for additional data file.Table S2Accession number for the protein sequences used in phylogenetic analyses.(TIF)Click here for additional data file.


As extensive experimental and computational efforts have been carried out to identify PKs and PPs, an integrative resource with detailed classification and annotation information would be of great value for both experimentalists and computational biologists. In this work, we first collected 1855 PKs and 347 PPs from the scientific literature and various public databases. Based on previously established rationales, we classified all of the known PKs and PPs into a hierarchical structure with three levels, i.e. group, family and individual PK/PP. There are 10 groups with 149 families for the PKs and 10 groups with 33 families for the PPs. We constructed 139 and 27 Hidden Markov Model profiles for PK and PP families, respectively. Then we systematically characterized ∼50 000 PKs and >10 000 PPs in eukaryotes. In addition, >500 PKs and >400 PPs were computationally identified by ortholog search. Finally, the online service of the EKPD database was implemented in PHP + MySQL + JavaScript.We present here EKPD ( As one of the most important post-translational modifications of proteins, the reversible phosphorylation is involved in a broad spectrum of biological processes ,2. Two tet al. comprehensively identified 130, 454, 240 and 518 putative PKs in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens, respectively  profile in Pfam, then narrowed down the candidates by motif scoring with a position-specific scoring matrix and ultimately performed a BLAST-based classification  ,11. In 2unctions . Based ounctions ,14. Receunctions ,16. The unctions ,16. In 2 , EnsemblPlants  and EnsemblFungi , respectively . More details on the classification of the PKs and PPs are provided in the Supplementary Results. Because the number of PKs and PPs is limited in several of the families, 139 and 27 HMM profiles were obtained for the PK and PP families, respectively. The catalytic domain sequences of the PKs and PPs were first aligned with MUSCLE , an extensively used tool for multiple sequence alignment (http://hmmer.janelia.org/) .Based on previously established rationales ,2,12–16,lignment . HMM modia.org/) . Furtheria.org/)  was sepaia.org/)  for eachia.org/)   . The prehttp://ekpd.biocuckoo.org/faq.php.For the families without any HMM profile, we conducted orthology searches  to identSupplementary Table S1 and S2). Although there are 395.5 PKs on average per animal, the average number of plant PKs is 3-fold (1202.0) higher than animals (Supplementary Table S1). A heatmap of the classifications and identification patterns for several of the major PK and PP groups was visualized using the ggplot2 program (http://had.co.nz/ggplot2/) in the R package (http://www.r-project.org/) (Supplementary Table S1). Also, there are 66.1 TK kinases on average per animal, whereas only 2.7 TK kinases on average were detected per plant (Supplementary Table S1). In addition, only a small proportion of PKs are TKL kinases (8.2%) in animals, whereas up to 60.4% of plant PKs were classified in the TKL group (Supplementary Table S1). This result is consistent with previous analyses, which have shown that TKL kinases predominantly occur in plants (Supplementary Table S2).In total, 50 433 PKs and 11 296 PPs from 84 eukaryotic species were identified, with an average of 600.4 PKs and 134.5 PPs per organism (ct.org/)   . From thct.org/) . For exan plants . The aveSupplementary Table S3) and PPs (Supplementary Table S4) were statistically analysed using a the hypergeometric test . Although a number of Pfam domains are preferentially over- or under-represented in both PKs and PPs, more domains are exclusively enriched or deprived in PKs or PPs (Supplementary Table S3 and S4). For example, SH2 (PF00017), which acts as a type of phosphotyrosine interacting domain (Supplementary Table S3 and S4). Meanwhile, the F-box domain (PF00646), which is found in specific proteins that recruit substrates for ubiquitination and proteasomal degradation (Supplementary Table S3 and S4). However, another type of phosphotyrosine-binding domain, PTB (PF08416) (Supplementary Table S4), whereas the SH3 domain (PF00018) (Supplementary Table S3). Furthermore, we statistically compared the different preferences of Pfam the domains in PKs (Supplementary Table S5) and PPs (Supplementary Table S6) in animals and plants using Yates' chi-squared (χ2) test . Interestingly, the SH2 domain occurs preferentially in both animal PKs and PPs compared with plants (Supplementary Table S5), whereas the SH3 domain preferentially occurs only in animal PKs (Supplementary Table S6).PKs and PPs are typically multidomain proteins containing a variety of other protein domains in addition to the catalytic domain. To identify the proteins domains that co-occur with the kinase and phosphatase catalytic domains, the distribution and diversity of the Pfam domain in the PKs   is only PF00018) , which iχ2) test  .The EKPD database was developed so as to be operable in an easy-to-use manner. Here we provide human protein kinase B (PKB or AKT1) as an example to illustrate the effective usage of EKPD. To make it easy to look through the data in EKPD, two approaches were implemented for the browse option: by species or by classification . In the e viewed A. As thee viewed A. Also, e viewed B. The lee viewed  and prese viewed B. Users e viewed B. By eite viewed A or the Akt page B, the meAkt page C. To orgAkt page C. The usAkt page D. More dThe identification and classification of PKs and PPs are fundamental for characterizing the regulatory roles of phosphorylation and dephosphorylation , predictS. cerevisiae, C. elegans, D. melanogaster, H. sapiens and A. thaliana , we obtained 2436 human phosphorylation sites modified by known PKs (Supplementary Table S7). Based on the identified site-specific kinase-substrate and phosphatase-substrate relations, we reconstructed a human kinase-phosphatase network, containing 62 PKs, 50 PPs and 87 common substrates (Supplementary Table S7). Furthermore, the S243 of transcription factor AP-1/c-Jun (JUN) is phosphorylated by GSK3A and dephosphorylated by PPP3CA, whereas the dephosphorylation regulates the c-Jun/Sp1 interaction (Supplementary Table S7).In eukaryotes, a protein substrate is phosphorylated by PKs and dephosphorylated by PPs ,39. The nown PKs . Also, wbstrates A. In parbstrates   ; Natural Science Foundation of China [31171263 and 81272578]; International Science & Technology Cooperation Program of China [0S2013ZR0003].Funding for open access charge: Conflict of interest statement. None declared.