C. crescentus is a dimeric histidine phosphotransferase that resembles the typical structure of histidine kinases.The cell-cycle regulator ChpT of  Caulobacter crescentus. ChpT is an essential histidine phosphotransferase that controls the activity of the master cell-cycle regulator CtrA by phosphorylation. Here, the 2.2 Å resolution crystal structure of ChpT is reported. ChpT is a homodimer and adopts the domain architecture of the intracellular part of class I histidine kinases. Each subunit consists of two distinct domains: an N-terminal helical hairpin domain and a C-terminal α/β domain. The two N-terminal domains are adjacent within the dimer, forming a four-helix bundle. The ChpT C-terminal domain adopts an atypical Bergerat ATP-binding fold.Two-component and phosphorelay signal-transduction proteins are crucial for bacterial cell-cycle regulation in  This essential cellular function is mainly controlled by two-component and phosphorelay system proteins , activation of CtrA depends on its phosphorylation at a conserved aspartate residue. Phosphorylation of CtrA is carried out by a phosphorelay composed of the membrane hybrid histidine kinase (HK) CckA  are usually monomeric proteins that form a typical structure named a four-helix bundle  is the residue that shuttles the phosphate, while the C-terminus weakly resembles an ATP-binding domain . Overexpression of His6-ChpT (25.29 kDa) was induced in Escherichia coli BL21 (DE3) cells at an OD600 nm of 0.6 by the addition of 100 µM isopropyl β-d-1-thiogalactopyranoside (IPTG) and cell growth was continued overnight at 293 K with shaking (215 rev min−1). The cells were harvested by centrifugation for 20 min at 500 rev min−1 and 277 K, resuspended in lysis buffer  and lysed using an Emulsiflex homogenizer  at 283 K. The supernatant containing His6-ChpT was purified using Ni2+–nitrilotriacetate (Ni–NTA) affinity resin  equilibrated with lysis buffer and was eluted with NPI-500 pH 8 . A second step of purification was performed by gel filtration using a HiLoad 16/60 Superdex 75 prep-grade column  equilibrated with running buffer . An SDS–PAGE of His6-ChpT purification is shown in Fig. 1a).The full-length DNA fragment of et al., 20076-ChpT was added to a final concentration of 56 µM in a 40 µl total reaction volume containing 5× SYPRO Orange dye and measurements were made using a Mx30005P real-time PCR instrument , raising the temperature from 298 to 368 K in 1 K intervals. The optimized buffer composition was 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 10%(w/v) glycerol.A thermal shift assay (TSA) was performed to optimize the buffer composition as described previously (Niesen 2.2.6-ChpT (0.5 mM) were performed by the sitting-drop vapour-diffusion method using the Cryos Suite kit . The best crystallization hit was obtained in condition No. 88, consisting of 24%(w/v) polyethylene glycol (PEG) 4000, 0.16 M MgCl2, 0.08 M Tris–HCl pH 8.5, 20%(w/v) glycerol. These conditions were subsequently refined using the hanging-drop vapour-diffusion method at 293 K in 24-well plates . Drops were prepared by mixing equal volumes (1 µl each) of protein solution and reservoir solution. 0.6 ml precipitant solution consisting of 15%(w/v) PEG 4000, 0.16 M MgCl2, 0.08 M Tris–HCl pH 8.5, 10%(w/v) glycerol was pipetted into the reservoir well. The crystal (obtained after 30 d at 293 K) used for data collection is shown in Fig. 1b).Initial crystallization trials of His6-ChpT was incubated at 293 K for 30 min in the presence of ATP or ADP (2.5 mM) and crystals were obtained under similar conditions.For crystallization trials with putative substrates, Hisw/v) in the presence of 100 mM Eu-DO3A  obtained from NatX-ray  as a cryoprotectant. The native and heavy-metal-derivative crystals were picked up in a nylon loop and flash-cooled  and europium-derivative (His6-ChpT-Eu-DO3A) crystals at 100 K on the PROXIMA1 beamline at the SOLEIL synchrotron  and BM30 at the European Synchrotron Radiation Facility , respectively. The data were processed with XDS  using SOLOMON  domain. The DHp domain contains the two H-boxes, with each catalytic His33 located on an opposite face of the four-helix bundle. Helix α1 extends for about 40 Å from the N-terminus to residue 50, displaying a kink induced by Pro38 at the end of the H-box . Helix α2 has a similar extension but without any pronounced kink. The four-helix bundle is stabilized mainly by hydrophobic interactions involving Leu27, Leu31, Phe35, Ala39, Ile42, Leu46, Met62, Ala69, Leu72 and Leu76.PDBeFold protein-structure comparison service at the European Bioinformatics Institute  domain is a close structural homologue of the ATP-binding domain of DesK   between strand β5 and helix α5 in ChpT, which together with the latter helix closes the ATP-binding pocket found in the CA domain of HKs. We also obtained crystals of His6-ChpT in the presence of 2.5 mM ATP or ADP in the crystallization buffer. Analyses of these crystals did not reveal any ATP or ADP bound to His6-ChpT, which was consistent with the different conformation of the CCT domain (data not shown).A structure-similarity search using the 6-ChpT homodimer shares the overall architecture of class I HK, with its four-helix bundle flanked by two ATP-binding-like domains, significant differences are observed in the relative orientation of these domains. In His6-ChpT the two domains adopt a compact conformation, with helices α3, α4 and α5 from the CCT domain lying along helices α1 and α2, leaving the five-stranded β-sheet of the CCT domain roughly parallel to the helical axis of helix α1. The KinB kinase from Geobacillus stearothermophilus (Bick et al., 2009et al., 2005Even if the HisThermotoga maritima (Marina et al., 2005et al., 2009et al., 2009et al., 2008et al., 2010et al., 2011via interactions with helix α1 of the DHp domain below the phosphodonor His residue and also parts of the ATP lid and the interdomain linker in the HKs. Since the ATP-lid region is severely affected in the ChpT structure by the N-terminal extension of helix α5 and the presence of an additional helix α4′, we anticipate that these differences may have implications for partner recognition of ChpT.A key characteristic of two-component systems is the high specificity of the HK–RR interaction (Laub & Goulian, 20076-ChpT, an essential phosphotransferase that controls the phosphorylation of CtrA in C. crescentus, adopts the class I histidine kinase fold, with a four-helix bundle flanked by two domains displaying a structurally different ATPase fold. This structure paves the way for future biochemical investigations aiming at deciphering the functional aspects of ChpT regulation and function.We have demonstrated in this work that HisChpT, 4fppPDB reference:


Plasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes. These exported proteins catalyse the formation of knobs on the erythrocyte plasma membrane and an overall increase in erythrocyte rigidity, presumably by modulating the endogenous erythrocyte cytoskeleton. In uninfected erythrocytes, Band 4.1 (4.1R) plays a key role in regulating erythrocyte shape by interacting with multiple proteins through the three lobes of its cloverleaf-shaped N-terminal domain. In P. falciparum-infected erythrocytes, the C-lobe of 4.1R interacts with the P. falciparum protein mature parasite-infected erythrocyte surface antigen (MESA), but it is not currently known whether other P. falciparum proteins bind to other lobes of the 4.1R N-terminal domain.P. falciparum infected erythrocytes.In order to identify novel 4.1R interacting proteins, a yeast two-hybrid screen was performed with a fragment of 4.1R containing both the N- and α-lobes. Positive interactions were confirmed and investigated using site-directed mutagenesis, and antibodies were raised against the interacting partner to characterise it’s expression and distribution in Yeast two-hybrid screening identified a positive interaction between the 4.1R N- and α-lobes and PF3D7_0402000. PF3D7_0402000 is a member of a large family of exported proteins that share a domain of unknown function, the PHIST domain. Domain mapping and site-directed mutagenesis established that it is the PHIST domain of PF3D7_0402000 that interacts with 4.1R. Native PF3D7_0402000 is localized at the parasitophorous vacuole membrane (PVM), and colocalizes with a subpopulation of 4.1R.P. falciparum exported proteins, including most members of the PHIST family, is unknown, and in only a handful of cases has a direct interaction between P. falciparum-exported proteins and components of the erythrocyte cytoskeleton been established. The interaction between 4.1R and PF3D7_0402000, and localization of PF3D7_0402000 with a sub-population of 4.1R at the PVM could indicate a role in modulating PVM structure. Further investigation into the mechanisms for 4.1R recruitment is needed.The function of the majority of PF3D7_0402000 was identified as a new binding partner for the major erythrocyte cytoskeletal protein, 4.1R. This interaction is consistent with a growing body of literature that suggests the PHIST family members function by interacting directly with erythrocyte proteins. Plasmodium falciparum parasites extensively remodel their host cells. As the parasite matures the erythrocyte becomes more rigid, loses its biconcave shape for a more spherocytic one, and small electron dense protrusions, known as knobs, form on the surface[Plasmodium falciparum achieves these massive changes in erythrocyte structure by exporting proteins into the erythrocyte cytosol, some of which interact with components of the host cytoskeleton and plasma membrane[P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the erythrocyte cytoskeleton through interactions with spectrin and actin[Intraerythrocytic  surface. These m surface,3. Plasmnd actin-6, and mnd actin,8.P. falciparum proteins that contain an export motif, referred to as the Plasmodium Export Element (PEXEL), little is know about their function[Plasmodium helical interspersed subtelomeric (PHIST) domain. PHIST domain-containing proteins can subdivided into three groups, PHISTa, PHISTb, and PHISTc, according to the position of one or two conserved tryptophans in the PHIST domain[For the majority of the more than 400 function,9,10. ThT domain. SeveralT domain. One PHIT domain, while aT domain but in t4.1R is involved in maintaining the biconcave shape, elasticity, and mechanical stability of human erythrocytes, and defects in 4.1R are one cause of hereditary erythrocyte elliptocytosis,16. 4.1RP. falciparum proteins also bind to it, and the C-lobe of the 4.1R FERM domain is recognized by MESA[P. falciparum proteins, a yeast two-hybrid screen of a P. falciparum cDNA library was performed using the N- and α- lobes of the 4.1R FERM domain as bait. This fragment of 4.1R interacted with PF3D7_0402000, an uncharacterized member of the PHISTa family. The 4.1R-PF3D7_0402000 interaction was mapped to the single amino acid level using the yeast two-hybrid system and visualized by immunofluorescence in P. falciparum-infected erythrocytes.Given the critical regulatory role that the 4.1R FERM domain plays in uninfected erythrocytes, it is not surprising that  by MESA,24. To tPlasmodium falciparum 3D7 strain parasites were cultured in O + human erythrocytes and serum (purchased from Interstate Blood Bank) as described previously[P. falciparum was approved by University of Alabama at Birmingham Institutional Review Board.eviously and syncP. falciparum cDNA library containing activation domain fusions with fragments of P. falciparum proteins was kindly provided by Dr Lawrence Bergman (Drexel University). The 4.1R bait plasmid was made by cloning a fragment of the FERM domain of 4.1R  into pGBKT7. The bait plasmid was transformed into yeast strain AH109, which was subsequently transformed with the P. falciparum cDNA-activation domain fusion library. Screening was performed by plating double transformants onto low stringency plates (−Leu/-Trp/-His) then replica plating onto high stringency plates (−Leu/-Trp/-His/-Ade) after four days growth. Activation domain plasmids were then rescued from all colonies that grew at high stringency, and retransformed into the original bait strain, as well as a strain transformed with an empty pGBKT7 vector, to test for plasmid independence and auto-activation.The Matchmaker GAL4 Two-Hybrid System 3 (Clontech) was used for yeast two-hybrid screens and analysis. A PF3D7_0402000 was cloned into pGADT7 . Once the 4.1R-PF3D7_0402000 interaction was confirmed, a series of deletion and point mutation constructs of PF3D7_0402000 were made. The positions of the deletions and mutations are described in the text, and were made using PCR primers as follows: ΔND1 construct , ΔCD1 construct , ΔND3 , PHIST (forward primer 5′- GGGAATTCATGTTAAGTCAGGACTATAATGAT-3′ reverse primer 5′-GGGGATCCTTAGTCTATAGGAATTTCCGTAG-3′). Subfragments of the PF3D7_0402000 PHIST domain were created as follows: PHISTΔC1 , PHISTΔC4 , PHISTΔC1C4 . Mutation of the conserved W residue within the PF3D7_0402000 PHIST domain was completed by PCR sewing . The PHIST domain from another PHIST family member was also cloned into pGADT7 as a control, using primers as follows: PF3D7_1253100 .In order to recapitulate the interaction observed in the yeast-two-hybrid screen, a fragment of All PF3D7_0402000 and PF3D7_1253100 constructs were cloned into pGADT7 and tested for interaction with the 4.1R bait plasmid in yeast strain AH109 at both medium (−Leu/-Trp/-His plates) and high stringency (−Leu/-Trp/-His/-Ade). Critical interactions were quantified by transforming the 4.1R bait and PF3D7_0402000 interacting plasmid into yeast strain Y187 (Clontech) and measuring β-galactosidase activity using the Pierce yeast β-galactosidase assay kit as directed by manufacturer instructions. All β-galactosidase assays were performed in triplicate.Escherichia coli BL21-DE3 pLysS. Recombinant proteins were expressed as hexa-his fusions and purified using either a Hi-Trap Chelating HP column (Amersham Biosciences) for the 4.1R fragment, or Nickel-NTA affinity agarose beads (Qiagen) for the PF3D7_0402000 fragment. The purified recombinant protein was then used to immunize both rabbits and rats . PF3D7_0402000 rabbit antisera was affinity purified by coupling the purified hexa-his fusion protein to Hi-Trap NHS-activated beads (Amersham Biosciences), and passing the rabbit antisera over a column containing the NHS-activated beads. After extensive washing with 1X PBS, affinity-purified antibodies were eluted from the column with low pH buffer  directly into a neutralization buffer  and dialysed for 24 hours against 1× PBS.A fragment of human 4.1R was cloned from cDNA into the expression vector pRSETB  and a fragment of PF3D7_0402000 was cloned into pRSETA . Both plasmids were transformed into P. falciparum-infected erythrocytes (1–3 × 108 parasites/ml) in a 1× SDS sample buffer with 5% β-mercaptoethanol. Triton X100 extracts were made by resuspending P. falciparum infected erythrocytes (3 × 108 parasites/ml) in an equal volume of 1% Triton X100 in PBS. After centrifugation, the supernatant was saved as the Triton soluble fraction and the pellet was washed three times in 1% Triton X100 in PBS. The pellet was resuspended in a 1× SDS sample buffer with 5% β-mercaptoethanol and labelled as the Triton X100 insoluble fraction. SDS lysates or Triton X100 extracts were loaded onto and run on 12% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were probed with primary antibodies as described, followed by HRP-conjugated secondary sera (anti-rabbit or anti-rat), and visualized using enhanced chemoluminescence .Parasite SDS lysates were made by resuspending P. falciparum were either directly probed with anti-4.1R, anti-PF3D7_0402000, and/or PfSERA5, or were fixed with an ice-cold 50% acetone/50% methanol solution and washed with 1× PBS before probing with the appropriate primary antisera. Secondary antisera (Molecular Probes) labeled with a fluorophore were used for visualization on an Olympus BX60 fluorescence microscope equipped with a Diagnostic Instruments Spot RT monochrome camera. All images were merged with Adobe Photoshop 5.0. Anti-PfSERA5[Air-dried thin smears of -PfSERA5 was a kiP. falciparum cDNA library . Screening was performed in the yeast strain AH109 and transformants were originally plated at low stringency (−Leu/-Trp/-His), then transferred to high stringency plates (−Leu/-Trp/-His/-Ade) after four days of growth. More than 1 × 106 colonies were screened and only nine grew on high stringency -Leu/-Trp/-His/-Ade plates. The plasmids from all nine positive colonies were isolated and retransformed with either the original 4.1R bait plasmid or an empty binding domain vector, pGBKT7, to test for plasmid independence and auto-activation. Two of the nine plasmids isolated from the screen did not recapitulate the growth on –Leu/-Trp/-His/-Ade plates and another three recapitulated the phenotype with the empty pGBKT7 vector, suggesting they were causing auto-activation.A fragment of the 4.1R FERM domain containing the N- and α-lobes was used as the bait in a yeast two-hybrid screen of a P. falciparum gene, PF3D7_0402000. Three of the four plasmids contained identical inserts , no growth was observed on Leu/-Trp/-His/-Ade– plates, suggesting this region is necessary for the PF3D7_0402000/4.1R interaction (amino acids 249–325). Deletion of an N-terminal fragment from the same plasmid, (ΔN1), also inhibited interaction with 4.1R, whereas a smaller N-terminal deletion, (ΔN3), allowed growth on both –Leu/-Trp/-His/and –Leu/-Trp/-His/-Ade plates, suggesting that this region is dispensable for interaction with 4.1R  was cloned into pGADT7 and tested for interaction with the 4.1R-bait plasmid. The PF3D7_0402000 PHIST domain alone was sufficient for interaction with 4.1R, as growth was observed on both –Leu/-Trp/-His and –Leu/-Trp/-His/-Ade plates  rather than primary sequence conservation. AlignmeTo test whether all four PHIST helices are necessary for PF3D7_0402000 binding to 4.1R, the first and fourth helices were deleted, both alone and together, from the PHIST binding domain construct. Deletion of either coil 1 or coil 4 of the PHIST domain individually (PHISTΔC1 and PHISTΔC4) or both helices together (PHISTΔC1C4), did not completely eliminate the interaction with 4.1R as all three constructs allowed growth on both –Leu/-Trp/-His and –Leu/-Trp/-His/-Ade plates , making the recovery of detectable amounts of 4.1R from detergent extracts by immunoprecipitation impossible. However, co-immunofluorescence with antibodies against PF3D7_0402000 and 4.1R show that a subpopulation of 4.1R clearly colocalizes with PF3D7_0402000 at the PVM  and P. falciparum infected (Pf) erythrocytes, with a major band at 80 kDa, the size of mature 4.1R  that is required for the export of many known exported proteins[P. falciparum-exported proteins that interact with the erythrocyte cytoskeleton and play a role in changing the structure of the erythrocyte membrane and cytoskeleton. The PHIST proteins, originally identified through searching the P. falciparum genome for PEXEL-containing proteins[in vivo.During the intraerythrocytic life cycle of proteins,10. The proteins, may plaproteins. In thisP. falciparum-infected erythrocytes,[P. falciparum-infected erythrocytes, divides 4.1R into two distinct populations: one that remains associated with erythrocyte cytoskeleton and one that dissociates from the erythrocyte cytoskeleton and is available to interact with PF3D7_0402000 at the PVM. This model is clearly hypothetical, but should be readily testable. Relocalization of erythrocyte cytoskeleton components to the PVM in P. falciparum-infected erythrocytes is not without precedent. Erythrocytes contain a class II myosin, which also appears to partially relocalize around the PVM (31). Moreover, it has been shown that 4.1R can bind to this class II myosin through its spectrin and actin-binding domain[How could only a subpopulation of 4.1R be recruited to the PVM? Interestingly, increased phosphorylation of 4.1R has been observed in rocytes,,32. Phosrocytes,. It is pg domain. Thus, iThe PVM is a dynamic membrane, which expands as the parasite divides and increases in size inside the erythrocyte. Given the role of 4.1R in maintaining structure at the erythrocyte surface, the presence of both 4.1R and myosin at the PVM is clearly consistent with a role in maintaining the stability of the PVM during this expansion process. It is also possible that 4.1R at the PVM plays a role in protein transport. Both the tubovesicular network, which consists of threadlike projections of the PVM that extend into the erythrocyte cytosol, and the formation of Maurer’s clefts, which may bud from the PVM,36, are P. falciparum parasite, it would seem reasonable to hypothesize that the PHIST domain has evolved to bind directly with an array of endogenous erythrocyte proteins, including components of the cytoskeleton, with individual PHIST domain proteins recognizing specific, and probably overlapping, targets. The PHIST family may therefore represent a central front in the parasite-host interactions that drive the successful colonization of human erythrocytes by P. falciparum parasites.The PHIST domain and PHIST family proteins were originally identified through bioinformatic approaches based on conserved structural features. The ideThe authors declare that they have no competing interests.LP, JR, DM, and MJ designed the experiments: LP, MJ, DM, and EK performed the experiments: LP, JR, DM, and MJ analysed the data: LP and JR wrote the paper. All authors read and approved the final manuscript.


Entamoeba histolytica is a protozoan parasite that infects humans and causes amebiasis affecting developing countries. Phagocytosis of epithelial cells, erythrocytes, leucocytes, and commensal microbiota bacteria is a major pathogenic mechanism used by this parasite. A Toll/IL-1R/Resistance (TIR) domain-containing protein is required in phagocytosis in the social ameba Dictyostelium discoideum, an ameba closely related to Entamoeba histolytica in phylogeny. In insects and vertebrates, TIR domain-containing proteins regulate phagocytic and cell activation. Therefore, we investigated whether E. histolytica expresses TIR domain-containing molecules that may be involved in the phagocytosis of erythrocytes and bacteria.in silico analysis we explored in Entamoeba histolytica databases for TIR domain containing sequences. After silencing TIR domain containing sequences in trophozoites by siRNA we evaluated phagocytosis of erythrocytes and bacteria.Using E. histolytica thioredoxin containing a TIR-like domain. The secondary and tertiary structure of this sequence exhibited structural similarity to TIR domain family. Thioredoxin transcripts silenced in E. histolytica trophozoites decreased erythrocytes and E. coli phagocytosis.We identified an E. histolytica could be an important element in erythrocytes and bacteria phagocytosis.TIR domain-containing thioredoxin of  Entamoeba histolytica is the etiological agent of amebiasis. It is estimated that this protozoan parasite infects 500 million people worldwide [E. histolytica. Phagocytosis requires recognition of ligands on target cells and activation of signaling pathways that lead to cytoskeletal reorganization and vesicle trafficking. In E. histolytica, the recognition of target cells is mediated by a galactose/N-acetylgalactosamine-binding lectin[er year). Phagocyg lectin and by ag lectin. This isg lectin.Dictyostelium discoideum, a Toll/IL-1R/resistance (TIR) domain-containing protein (TirA) is required for the phagocytosis of bacteria, which is essential for nutrition and for protection against infection[Phagocytosis is essential for the survival of unicellular organisms. In the social ameba nfection. Moreovenfection.E. histolytica in phylogeny) to insects and vertebrates. It has been suggested that in E. histolytica, the phagocytosis of natural substrates (such as human erythrocytes and E. coli) is mediated by TIR domain-containing proteins that participate in a signaling component that existed before the diversification of eukaryotes[E. histolytica expresses TIR domain-containing molecules that participate in the regulation of the phagocytosis of human erythrocytes and E. coli. Using an in silico analysis of the E. histolytica proteome, we identified a TIR domain-containing sequence that corresponds to a thioredoxin. Furthermore, the downregulation of this thioredoxin by siRNA led to decrease of phagocytosis of erythrocytes and E. coli by E. histolytica trophozoites. These results suggest that the TIR domain-containing thioredoxin is involved in E. histolytica phagocytosis.The TIR domain is present in a wide variety of eukaryotic organisms, from free-living amebas (which are closely related to karyotes. Therefohttp://hmmer.janelia.org) and a seed alignment collection of TIR proteins from PFAM database. [http://www.sanger.ac.uk), and the Pathema Bioinformatics Resource Center (http://pathema.jcvi.org) databases for Entamoeba genus. Using BLAST  on E. histolytica proteins of NCBI databases, sequences of primary structure scoring E values < 0.001 with TIR domain-containing proteins of Entamoeba species were selected as homologous proteins.A Hidden Markov Model (HMM) for TIR domain proteins was built with HMMER software v2.3.2 (re/Pfam). ThroughEntamoeba histolytica identified were compared with the primary structures of the TIR domains of Arabidopsis thaliana , Drosophila melanogaster , and Homo sapiens  by a multiple sequence alignment calculated with T-Coffee (http://tcoffee.crg.cat/). Secondary structures were calculated with the Psipred Protein Structure Prediction Server (http://bioinf.cs.ucl.ac.uk/psipred/). The tertiary structure of the TIR domain of the identified protein was modeled using I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/[http://www.cgl.ucsf.edu/chimera/). The obtained structures were displayed in pdb format using RasMol v. 2.6.The primary and secondary structures of the TIR domain-containing proteins of I-TASSER/,18) and E. histolytica strain HM-1:IMSS were axenically grown in TYI-S-33 medium, according to Diamond et al.[6 cells/mL.Trophozoites of the d et al.. TrophozE. histolytica trophozoites using the TRIzol reagent . RNA was treated with DNase  and reverse-transcribed using SuperScript II RNase H-Reverse Transcriptase . Primers for thioredoxin and PATMK (which was used as an expression control) were designed using Primer3 v.0.4.0 (http://frodo.wi.mit.edu/primer3[2 (Promega), 0.25 μl of dimethyl sulfoxide , 2.5 U of Taq DNA polymerase (Promega), 0.5 μM of each primer , annealing (59°C for 90 s), and extension (72°C for 90 s); a final extension of 5 min at 72°C was also performed. PCR products were electrophoresed in 2% agarose gels at 70 V for 60 min and stained with GelRed™ Nucleic Acid Stain (Invitrogen). The density band was documented by a EC3 Bioimaging System , using ultraviolet illumination. Densitometric analysis was made using VisionWorksLS program v6.4.3 .Total RNA was extracted from u/primer3) and actu/primer3. The finhttp://www.idtdna.com/Scitools/Applications/RNAi/RNAi.aspx). For each gene, three double-stranded oligonucleotides of 21–23 bp that bound to the initial, mid, and terminal regions of the corresponding transcripts were used. Each oligonucleotide  were added to 106 trophozoites in a final volume of 1.0 mL of TYI-S-33 medium, and the trophozoites were cultured for 15 h at 37°C . The sig for 20 min, to remove mononuclear cells. Erythrocytes were washed and suspended in phosphate-buffered saline solution. Erythrocytes (1 × 107) were incubated with 1 × 106 trophozoites (untreated or in the presence of thioredoxin or PATMK silencing) in 1 mL of TYI-S-33 medium at 37°C for 30 min, with agitation at 150 rpm. One milliliter of phosphate-buffered saline solution was added after the incubation, and the culture plate was centrifuged at 600 × g for 5 min. The pellet was lysed in 1 mL of cold concentrated formic acid (Sigma-Aldrich), and absorbance at 400 nm was measured using a spectrophotometer . Results were analyzed using Repeated-measures one-way ANOVA and Bonferroni test. Significance was set at P < 0.05.All experimental procedures were approved by the Local Ethical Committee of Health Research of Specialties Hospital, Mexican Social Security Institute (IMSS), Mexico City, Mexico (Register number. R-2009-3601-157). Venous blood (2 mL) from healthy donors was layered over Lymphoprep™  and centrifuged at 800 × Escherichia coli strain DH-5α was transformed with a plasmid encoding GFP and an ampicillin resistance gene and grown overnight at 37°C in LB medium with 100 mg/mL of ampicillin. Bacteria were harvested by centrifugation at 9,000 × g for 10 min, washed three times with phosphate-buffered saline solution, and suspended in phosphate-buffered saline solution to a concentration of 1 × 108 colony forming unit (CFU) per mL. Incubation of 1 × 108 CFU with 1 × 106 trophozoites (untreated or in the presence of thioredoxin or PATMK silencing) was performed in 1 mL of TYI-S-33 medium at 37°C for 45 min, with agitation at 150 rpm. Gentamicin (15 ng) was added after the incubation; 10 min later, the culture plate was centrifuged at 600 × g for 5 min. The pellet was suspended in 1 mL of phosphate-buffered saline solution, and the fluorescence of each sample was measured using a flow cytometer . At least 10,000 events were acquired per sample. Data were analyzed using BD FACS DiVa software 2.1, and mean fluorescence intensity (MFI) was reported. Results were analyzed using Repeated-measures one-way ANOVA and Bonferroni test. Significance was set at P < 0.05.The Entamoeba genus. Two hypothetical proteins  and two putative proteins  were identified. A exploration on Entamoeba histolytica Protein databases for homologues TIR-domain containing proteins of any Entamoeba genus led us to identify a sequence which also contain a thioredoxin domain (GenPept:XP_649779). This whole sequence will be termed “thioredoxin” henceforth.We generated a HMM from a seed alignment (Pfam:PF01582) of TIR domain and searched proteins containing a TIR domain in Pathema database for D. melanogaster Toll4, and A. thaliana TAO1  . Both TIR domains, of mammalian IL-1R and TLR2, mediate homotypic protein–protein interactions in the signal transduction that facilitate the activation and expression of mechanisms which increase several biological process, including phagocytic activity[The TIR domain consists of approximately 200 amino acids that form five β sheets surrounded by five α helixes. In anim helixes. On the activity,28,29.Dictyostelium discoideum[Escherichia coli) and erythrocytes to uptake iron[E. histolytica, phagocytosis process is Gal/GalNAc lectin and PATMK dependent[The participation of this domain on phagocytosis during infection resistance and feeding observed in the evolutionary related ameba iscoideum,15,30, lake iron. In E. hependent.E. coli after incubation with small interfering double stranded-RNAs for thioredoxin, suggesting a role for the TIR domain-containing thioredoxin in trophozoites phagocytosis. These results could support the proteomic approach explored by McCoy et al., and Okada et al.[In this report, we observed reduction in phagocytosis of human erythrocytes and da et al.,33, whicda et al.-10. If tThe above mentioned, is similar to reported to the majority of these TIR domain-containing proteins that have been described in plants, insects, and vertebrates and participate in phagocytic and cell activation,25. HoweE. histolytica expresses other proteins with thioredoxin activity that participate in nicotinamide adenine dinucleotide phosphate (NADPH)-dependent hydrogen peroxide reduction[A diverse number of specific functions and cofactor activities have been described for thioredoxins. These functions include growth factor activity, cell protection from toxic compounds , activation of inflammatory pathways, regulation of transcription factors and protein binding, and catalysis of protein folding; each of these activities has an effect on cellular responses to toxic insults,12. Howeeduction; these dE. histolytica thioredoxin (GenPept:XP_649779) has a TIR-domain. Considering that several members of TIR domain family have been involved in signal transduction in many organisms, and silencing its expression decrease erythrocytes and bacteria phagocytosis, the thioredoxin could play a central role in signal transduction during this event, hence influencing the E. histolytica survival mechanisms.In this report we present evidence that protein sequence corresponding to 344–442 position in Entamoeba histolytica by phagocytosis involves recruitment of proteins such as PATMK, and our results suggest that TIR domain-containing thioredoxin also can support this activity on bacterial and erythrocytes phagocytosis.Uptake of nutrients in The authors have declared that no competing interests exist.IMH, AMT, AJU, IWB, and CLM conceived the study, and all authors participated in the study design, coordination and writing the manuscript. All authors read and approved the final manuscript.Table S1. Double-stranded oligonucleotides used for the silencing of the expression of thioredoxin and PATMK in E. histolytica trophozoites.Click here for file


Interestingly, whilst the WW domains and TEAD-binding domain are highly conserved in the Drosophila melanogaster YAP orthologue, Yorkie, the majority of the C-terminal region of YAP is not present in Yorkie. To investigate this apparent conundrum, we assessed the functional roles of the YAP and Yorkie C-termini. We found that these regions were not required for Yorkie's ability to drive tissue growth in vivo, or YAP's ability to promote anchorage-independent growth or resistance to contact inhibition. However, the YAP transactivation domain was required for YAP's ability to induce cell migration and invasion. Moreover, a role for the YAP transactivation domain in cell transformation was uncovered when the YAP WW domains were mutated together with the transactivation domain. This shows that YAP can promote cell transformation in a flexible manner, presumably by contacting transcriptional regulatory proteins either via its WW domains or its transactivation domain.The Salvador-Warts-Hippo (SWH) pathway is an important regulator of tissue growth that is frequently subverted in human cancer. The key oncoprotein of the SWH pathway is the transcriptional co-activator, Yes-associated protein (YAP). YAP promotes tissue growth and transformation of cultured cells by interacting with transcriptional regulatory proteins via its WW domains, or, in the case of the TEAD1-4 transcription factors, an N-terminal binding domain. YAP possesses a putative transactivation domain in its C-terminus that is necessary to stimulate transcription factors  Drosophila melanogasterD. melanogaster and mammals, and deregulation of the pathway leads to egregious organ overgrowth D. melanogaster, core components of the SWH pathway include the scaffold proteins, Salvador (Sav) and Mob as tumor suppressor (Mats), and the S/T kinases, Warts (Wts) and Hippo (Hpo). These proteins limit organ growth by phosphorylation-mediated inhibition of Yorkie (Yki), which is homologous to YAP in mammals D. melanogaster Wts) phosphorylate YAP on five sites, of which S127 and S381 appear to be the most important The Yes-associated protein (YAP) is a transcription co-activator that mediates the transcriptional output of the Salvador/Warts/Hippo (SWH) pathway, which is a tumour suppressor pathway that was first identified in D. melanogaster tissues, several points of evidence have shown that YAP has oncogenic potential in mammals. Overexpression of YAP can confer anchorage-independent growth of NIH3T3 or MCF10A cells and can stimulate growth-factor independent growth, migration and invasion of MCF10A cells, which are hallmark properties of oncogenes YAP gene was found to be amplified in mouse models of breast and liver cancer Following the discovery that Yki promotes the growth of D. melanogaster, the sole TEAD1-4 homologue, Scalloped (Sd), interacts with Yki and is required for Yki-induced tissue overgrowth Although the mechanism of YAP-induced oncogenesis is not fully understood, several studies have suggested that the TEAD1-4 transcription factors are major mediators of YAP's growth-promoting ability. YAP activates TEAD1-4 and stimulates transcription of known TEAD1-4 target genes As well as possessing a TEAD1-4-binding region in its N-terminus, YAP possesses several other conserved protein domains. It harbors two WW domains that mediate interactions with important regulatory proteins that either promote or inhibit YAP's ability to drive transcription and promote cell transformation, migration and epithelial to mesenchymal transition in a cell- and context-dependent fashion in vivo. Surprisingly, we found that the TA domain was dispensable for YAP-mediated transformation and proliferation. By contrast, this domain was required for YAP to stimulate cell migration and invasion. These results question an obligate requirement for the putative TA domain for YAP's ability to act as a transcriptional co-activator protein.Therefore, we sought to investigate the requirement of the TA domain for YAP's ability to stimulate cell proliferation, migration, invasion and oncogenic transformation, as well as the C-terminus of Yki for its ability to stimulate tissue growth UAS-yki or UAS-ykiΔC transgenes  and imaged on an Olympus BX-51 microscope. Wing size was quantified using Adobe Photoshop CS3 as in B5yki mutant clones (with and without UAS-yki transgene expression) were generated by a 15 min heat shock 72 hrs after egg deposition and were dissected 49 hrs later.Imaginal wing discs were stained as described MCF10A and NIH-3T3 cell transfection and infection were performed as described previously YAP-S127A, YAP-WW1+2* and YAP-S94A mutations were described previously CCGGATCCACCATGGACTACAAAGACGATGACGACAAGATGGACCCCGGGCAGCAGYAP-F: CCGAATTCCTAAGCACTCTGACTGATTCTCYAP-ΔC-R: CCGAATTCCTATAACCATGTAAGAAAGCTTTCTGGGCTCTGGGGAGCCAGYAP-ΔTA-R: CCGAATTCCTATAACCATGTAAGAAAGCTTTCAGCACTCTGACTGATTCTCCCGAATTCATGGAACAAAAACTCATCTCAGAAGAGGATCTGTGCGCGTGCCTAATCYki-Myc-F: CCTCTAGATTATTGCATTCTGGGATCATTCCYki- ΔC-R: D. melanogaster Yki displays strong conservation of the WW and TEAD-binding domains, it is bereft of the majority of C-terminal sequences found in YAP and has only 55 amino acids following the second WW domain compared with 242 amino acids in YAP2L. Despite this, it is still formally possible that the shorter Yki C-terminus possesses the ability to regulate transcription factor activity and tissue growth.Homologues of YAP are present in many species from holozoans to humans UAS-inducible yki transgenes; one encoding a full length Yki protein and one encoding a Yki protein lacking the last 51 amino acids (Yki-ΔC) . To determine whether the C-terminus of Yki was required for Yki to regulate ex transcription, we assessed ex levels using a lacZ enhancer-trap in the ex locus in the posterior compartment of the wing. In the presence of either yki transgene, lacZ levels were elevated to similar degrees, showing that Yki does not require its C-terminus to regulate transcription from the ex locus  . Each trergrowth . To verisue type . Yki driex locus . Finally degrees . CollectYAP2L or derivatives of the YAP2L gene, but for simplicity, are referred to as YAP. Two mutants lacking either the entire C-terminal domain following the second WW domain, YAP-ΔC (deletion of residues 278 to 504 in YAP2L), or the region previously defined as the YAP transactivation domain The ability to induce anchorage-independent cell growth is a hallmark of oncogenes; this includes YAP, which has been shown to promote anchorage-independent growth of both MCF10A and NIH-3T3 cells To assess the ability of different YAP mutants to confer anchorage-independent cell growth, each cell line was plated in soft agar assay and incubated for 7 (NIH-3T3) or 14 (MCF10A) days. Consistent with previous reports, YAP induced robust colony growth in soft agar compared with the vector control. By contrast, YAP-ΔC failed to induce growth in soft agar . SimilarTEAD1-4 transcription factors are major effectors of the oncogenic activity of YAP D. melanogaster orthologue, Yki, played cell-specific roles with respect to induction of cell transformation and tissue growth, respectively Previously, we discovered that the WW domains of YAP and its In previous studies, YAP was shown to enhance the proliferation rate of both NIH-3T3 cells grown in a two-dimensional (2D) culture, as well as MCF10A cell colony size grown in a three dimensional (3D) matrigel assay In addition, we assessed the size of acini formed by MCF10A cells that expressed different YAP variants, in 3D matrigel. As shown in To investigate the role of the TA domain of YAP in other functional settings, we assessed the ability of each YAP mutant to promote invasive growth in a 3D matrigel assay. Consistent with previous reports, overexpression of YAP-S127A in MCF10A cells cultured in matrigel caused spike-like protrusions, which is defined as an invasive phenotype [data not shown and in vitroThe Yorkie and YAP transcriptional co-activator proteins are conserved regulators of tissue growth in flies and mammals, respectively. Given the high degree of functional conservation between these proteins and the mechanism by which they are regulated, we were intrigued by the lack of conservation in the C-termini of these proteins, particularly because the C-terminus of YAP contains a transactivation domain that is necessary for its ability to activate transcription factors Based on the previous finding that YAP possessed a potent TA domain that was required for YAP to activate the pEBP2α transcription factor D. melanogaster.By contrast, the TA domain was required for YAP to stimulate cell migration and invasive properties in cells cultured in 3D. This suggests that the YAP TA domain has context-specific roles: it is required for YAP to regulate genes that stimulate migratory behaviour, but not proliferation and survival. We previously observed a similar relationship when studying WW domain mutant versions of YAP in NIH-3T3 cells. A hyperactive version of YAP required its WW domains to stimulate cell migration, but not to promote growth in soft agar Interestingly, when either the WW domains or TA domain were mutated, YAP's transformation potential increased substantially, but when both domains were mutated, YAP activity was lost. This suggests that YAP is a flexible transcription co-activator and that it can regulate transcription of genes that transform cells by complexing with proteins either through its WW domain or its TA domain. In either scenario, we found that YAP stimulates transformation via TEAD transcription factors. Our finding that the TA domain is not required to mediate transformation via TEAD transcription factors is supported by our study of Yki. The WW domains and the TEAD-binding domain are conserved between Yki and YAP, but the Yki and YAP C-termini are poorly conserved. The fact that a Yki protein that lacked the C-terminus could rescue loss of the wild-type protein shows that Yki activates its partner transcription factors independently of its C-terminus. Based on several recent studies, Yki most likely interacts with transcriptional regulatory proteins via its WW domains The SWH pathway has been reported to be frequently subverted in human cancer, largely based on the observation that YAP is localized to the nucleus in a high percentage of solid tumours. Based on these findings and YAP's potent pro-tissue growth and pro-transformation activity, it has been touted as a candidate for therapeutic intervention. This study highlights a remarkable degree of complexity in both the regulation of YAP activity and the mechanism by which YAP regulates gene transcription. It also suggests redundant mechanisms by which YAP can regulate expression of genes that promote cell transformation, and that the same YAP domains mediate both positive and negative regulatory interactions. Therefore, great care will be required when designing therapies aimed at disabling YAP function in transformed cells.Figure S1Expression of wild-type and mutant YAP proteins in MCF10A and NIH-3T3 cells. Expression levels of YAP in cells stably expressing vector alone (CON) or various YAP proteins in either MCF10A or NIH-3T3 cells. Actin levels were determined to ensure even loading. Molecular mass markers in kDa are shown on the left.(TIFF)Click here for additional data file.


The Baltic Sea is characterized by hyposaline surface waters, hypoxic and anoxic deep waters and sediments. These conditions, which in turn lead to a steep oxygen gradient, are particularly evident at Landsort Deep in the Baltic Proper. Given these substantial differences in environmental parameters at Landsort Deep, we performed a metagenomic census spanning surface to sediment to establish whether the microbial communities at this site are as stratified as the physical environment. We report strong stratification across a depth transect for both functional capacity and taxonomic affiliation, with functional capacity corresponding most closely to key environmental parameters of oxygen, salinity and temperature. We report similarities in functional capacity between the hypoxic community and hadal zone communities, underscoring the substantial degree of eutrophication in the Baltic Proper. Reconstruction of the nitrogen cycle at Landsort deep shows potential for syntrophy between archaeal ammonium oxidizers and bacterial denitrification at anoxic depths, while anaerobic ammonium oxidation genes are absent, despite substantial ammonium levels below the chemocline. Our census also reveals enrichment in genetic prerequisites for a copiotrophic lifestyle and resistance mechanisms reflecting adaptation to prevalent eutrophic conditions and the accumulation of environmental pollutants resulting from ongoing anthropogenic pressures in the Baltic Sea. The Baltic Sea is the world’s second largest body of brackish water, sustained by inflow of freshwater from the surrounding drainage area and only occasional inflow of oxygen-rich saltwater from the North Sea via the Danish straits Stratification of microbial community structure and functional capacity has been observed in a range of marine environments Hypoxia is another defining feature of the Baltic Sea, and has been a periodic feature since its formation st century The Baltic Sea has a large catchment, and a further major environmental problem, anthropogenic pollution, is well documented. Long-lived pollutants, such as heavy metals and polyaromatic hydrocarbons (PAHs), are known to accumulate in sediments. While levels of some organic pollutants such as DDT are reducing We sought to establish the degree to which microbial communities with their functional capacities are stratified in the Baltic Proper. Our chosen study site was Landsort Deep, which, as the deepest point in the Baltic Sea, is minimally affected by occasional inflows, leading to sustained stratification, with acute anoxia below the halocline Metagenomes were generated from a bottom sediment sample and three water column samples across a depth transect spanning Landsort Deep surface water (10–20 m), the mixed layer (70–80 m), anoxic water (400–410 m) and bottom sediment (466 m). The environments from which each of the four metagenome samples were derived showed marked differences in key environmental parameters, including salinity, temperature, light and concentrations of dissolved oxygen, nitrate, ammonium, sulphide and phosphorous. Of particular note is the pronounced oxygen stratification across the study transect from surface water through to the bottom sediment .The metagenomes of the four microbial communities sampled generated approximately 417 Mb of 454 GS FLX Titanium sequencing data consisting of 1 205 630 sequence reads, with an average sequence length of 344 bp, after removal of duplicate sequence reads using CDHIT-454 . For eacThe triplicate sediment samples showed similar taxonomic and functional profiles as shown by low Bray-Curtis distances . SimilarGiven that the four environments across the depth transect showed substantial differences in environmental parameters , we soug3− is available With evident microbial community stratification, we next examined how this was reflected in the N-dependent and the S-dependent functional capacities, respectively, across the Landsort Deep metagenomic transect. The Baltic Proper is characterised by excessive loading of nutrients and a ‘vicious circle’ of feedbacks that exacerbate eutrophication and drive phytoplankton blooms 2−/NO3− concentrations were low (2− and NO3−) through nitrification at oxic conditions Sulfurimonas gotlandica GD1 at 400 m  in the mixed layer and anoxic water at Landsort Deep. This co-distribution of sulphur-oxidizing epsilonproteobacteria and ammonium oxidizing Thaumarchaeota is indicative of a syntrophic interaction.We find that the distribution of Epsilonproteobacteria largely coincides across the Landsort Deep transect (Pearson’s r = 0.83) with sulphide insensitive Thaumarchaeota http://nest.su.se/). We therefore assessed if the increased availability of ammonium coincides with nitrogen removal through anaerobic oxidation of ammonium (anammox). Anammox bacteria related reads (Candidatus Kuenenia stuttgartiensis) were identified primarily in the anoxic water (0.3%) and sediment (0.3%) but no hydrazine oxidase genes were detected. At the time of sampling we therefore find no capacity for anammox at Landsort Deep. While this may reflect finite sampling, we note that this result is consistent with another recent study where no anammox activity was reported for Landsort Deep in spring 2009, despite anammox being detected at the hypoxic/anoxic zone of Gotland Deep Over the past decade the concentration of ammonium, as a product of organic matter mineralization, has drastically increased below the halocline at Landsort Deep , indicating that a role for DNRA, at the time of sampling would be limited.Nitrogen might also be prevented from being removed, and instead recycled or stored in the ecosystem at reduced conditions, potentially increasing the eutrophication effect through anaerobic dissimilatory reduction of nitrate to ammonia . We detected only few nifHDK and nifENB) and found clear stratification of this process, at subsystem level, across Landsort Deep communities, with an increased capacity at depth  sulphate-reducing and naphthalene degrading Deltaproteobacteria NaphS2 Fixation of nitrogen is at least as important process as nitrogen removal. We detected the minimum gene set required for nitrogen fixation , with samples derived from water column, sediment, soil and compost.To generate a quantitative measure of similarity, we made use of phylogenetic network methods implemented in MEGAN 4 Highly similar taxonomic profiles are also evident between sediment and 400 m water column communities at Landsort Deep  and S3. The taxonomic profile of the surface water community at Landsort Deep groups closely with three surface water metagenomes from the Western English Channel. Similarities between these metagenomes include a significant (q<0.05) overrepresentation of Rhodobacteraceae, Sphingobacteriaceae, unclassified Methylophilales and unclassified Flavobacteria, the three latter potentially due to high loads of organic particles Of the Landsort Deep communities, the 75 m community, from the oxic-anoxic interface is an outlier when it comes to taxonomy, as it falls slightly outside the main groups that emerge in this analysis, the closest being surface water at Landsort Deep and Western Channel. The 75 m Landsort metagenome derives from a brackish environment with a steep biogeochemical gradient, so this community would not be expected to share similar taxonomical composition with open ocean communities of similar depths Our CA results  indicateAlthough functional capacity varies greatly across the Landsort Deep transect we observe that the metagenomes as a group, when compared against all other metagenomes, are significantly  underrepresented in genes associated with biotin biosynthesis but enriched in functional capacities (SEED hierarchy 3) of osmoregulation and metabolite detoxification (glutathione-regulated potassium-efflux system protein KefC), transcriptional regulation (molybdate-binding domain of ModE) of several genes of biogeochemical pathway enzymes including dimethyl sulfoxide reductase, nitrate reductase and hydrogenases, and motility (flagellum), several possible indicators of a stratified environment.Another probable indicator of eutrophication at Landsort Deep is the significant  overrepresentation of Flavobacteriaceae (7.4%)  in surfaCarbohydrate utilisation was also evident in sediment at Landsort Deep . Genes fThe impact of pollution at Landsort Deep was clearly reflected by a comparatively high abundance of resistance genes for cobalt, cadmium and zinc in the 400 m and sediment communities  and S6a.Another probable sign of adaptation to a polluted environment is the comparatively high abundance of integrons in all Landsort Deep metagenomes , in partThe unique conditions of the Baltic Proper combined with infrequent turnover of deeper waters have led to strong stratification of the water column. Our survey at Landsort Deep, the deepest point in the Baltic Sea, is the first metagenomic survey of any deep (>400 m) water or sediment of the Baltic Sea. It clearly shows strong stratification of both microbial taxa and functional capacities across a depth transect spanning from surface water, through the oxycline, into the anoxic deep waters and sediment. Significantly, functional capacity corresponds more closely to environment than does taxonomic affiliation, suggesting that a range of taxa may be using very similar functional gene repertoires to adapt to the considerable environmental constraints present across our sample site. As predicted given known physical parameters, we find a marked effect of the coupled parameters salinity, temperature and oxygen content on the microbial community. The unique oceanographic characteristics of the Baltic Proper, the bathymetry and the impact this has on nutrient dynamics and the distribution of pollutants, is reflected in the Landsort Deep microbial communities that are characterized by gene repertoires shaped by both anthropogenic pollution and eutrophication. The present study clearly documents this influence on Baltic Sea microbial communities at Landsort Deep.Sampling was conducted within the Stockholm Marine Research Centre  at monitoring station BY31. The station is not a national park, private land or protected area. No specific permissions were required. The study did not involve endangered or protected species.Water and sediment samples were collected on 15 April 2009 during the spring bloom at Landsort Deep  in the Baltic Sea, Sweden. Triplicate sediment cores for DNA extraction and elemental analysis were retrieved from 466 m depth using a Kajak sampler. Concomitantly, salinity, dissolved oxygen concentration, temperature and pH were measured in the water phase as close to the sediment surface as possible. Pore water was extracted from the sediment top layer (0–10 cm) using Rhizon Soil Moisture Samplers . Water (30 L) was sampled from three distinct zones, the surface water at 10–20 m, the mixed layer at 70–80 m and the anoxic zone at 400–410 m depth, using 5 L Niskin bottles. Samples were pre-filtered through 3 µm 47 mm polycarbonate filters  onto 0.22 µm Sterivex GS filters  that were immediately immersed in lysis buffer. Filtrates and sediment samples were frozen on board, transferred on dry ice to the laboratory and stored at −80°C, until DNA extraction.Nutrient data were determined and provided by the Department of Systems Ecology at Stockholm University.Total DNA was extracted from the sediment top layer (0–10 cm) in 200 mg aliquots, using the FastDNA® SPIN Kit for Soil  and total DNA of filters from water samples was extracted according to Duplicate sequence reads that passed standard GS FLX quality criteria removed with CDHIT-454 CA of taxa  and functional capacity , based on abundance data normalised to 100000 reads per dataset, were performed in R Landsort Deep data was compared with other metagenomes including marine metagenomes from the Marmara Sea Figure S1Pairwise statistical analyses (n = 6) of sequence reads assigned to SEED categories (hierarchy 2) in the four Landsort Deep metagenomes. Only statistically significant differences , where the SEED categories are represented with >100 reads in at least one metagenome, are shown.(PDF)Click here for additional data file.Figure S2Relative abundance of sequence reads assigned to phyla (or classes in the case of Proteobacteria) within the bacterial/archaeal communities. Only taxa with >1% of the assigned sequence reads in any of the four Landsort Deep communities were included.(PDF)Click here for additional data file.Figure S3Comparative analysis of the taxonomic distribution in Landsort Deep communities with that of other aquatic metagenomes based on relative abundance of taxa at the family rank of the NCBI taxonomy in MEGAN. Hierarchical clustering was generated with Kendall’s Tau distance metric and average linkage clustering.(PDF)Click here for additional data file.Figure S4Pairwise statistical comparison of functional capacities (SEED hierarchy 1) between cluster of metagenomes. The cluster  of metagenomes of Landsort Deep 400 m and sediment, Marmara Sea sediment and Tonya Seep sediments was compared to all other marine metagenomes using Welch’s t-test and Storey’s FDR (q<0.05) in STAMP.(PDF)Click here for additional data file.Figure S5Comparative analysis of functional capacity of Landsort Deep communities with that of other aquatic metagenomes based on relative abundance of SEED categories (hierarchy 3). Yellow to black colour represents high and low relative abundance of SEED categories. Hierarchical clustering was performed using Kendall’s Tau distance metric and average linkage clustering.(PDF)Click here for additional data file.Figure S6Relative abundance of sequence reads assigned to the SEED categories Cobalt zinc cadmium resistance (a) and Integrons (b), respectively, in marine metagenomes. Clustering of metagenomes is based on results of the comparative network analysis . Horizon(PDF)Click here for additional data file.Table S1th of April concurrently with sampling of water and sediment for metagenomic analyses.Environmental parameters collected at Station BY31, Landsort Deep, Baltic Sea at the 15(PDF)Click here for additional data file.Table S2Summary of sequence and annotation data for the four Landsort Deep metagenomic data sets. Taxonomical and functional assignments of sequence reads were performed with MEGAN 4.(PDF)Click here for additional data file.Table S3Ecological distances between the Landsort Deep metagenomes. Dissimilarity indices were calculated with Bray-Curtis distance metric at the taxonomic level of family.(PDF)Click here for additional data file.Table S4Correlation between environmental parameters and distribution of functional capacities and taxa, respectively. Pearson’s correlations were performed based on coordinates of SEED categories (hierarchy 2) and taxa (family rank) along the three ordination axes from correspondence analysis.(PDF)Click here for additional data file.Table S5Species scores (coordinates) from CA for the different taxa (at the rank of family) and SEED categories (hierarchy 2), respectively. Shown are also abundances in each sample normalised to 100 000 reads per dataset. Taxa and SEED categories are ordered from low to high CA1 coordinates.(PDF)Click here for additional data file.Text S1Supporting material and methods.(PDF)Click here for additional data file.


CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. Low-resolution shape reconstructions, obtained from small angle X-ray scattering, suggest that the tight cooperation between the domains is mediated by intramolecular interactions due to their spatial proximity within CHD4. We present a plausible regulatory mechanism necessary for the nucleosome remodelling activity of CHD4.22.1Escherichia coli BL21 (DE3) cells. Expression in LB or TB media was induced with 0.7 mM of isopropyl-thiogalactopyranoside (IPTG) when culture reached OD600 = 0.6 and incubated overnight at 20 °C. Cells were lysed in 50 mM sodium phosphate pH 7.5, 500 mM NaCl, 5 mM imidazole, 0.2% Tween 20, protease inhibitor cocktail tablets (Roche) and the obtained supernatant mixed with TalonTM resin (Clontech). The recombinant proteins were eluted with imidazole gradients and further purified by size exclusion chromatography in 20 mM Tris pH 7.5, 200 mM NaCl, 1 mM DTT.C-terminal 8xHis-tagged constructs were generated by PCR using human CHD4 cDNA  as a template and appropriate sets of primers. The amplified PCR products were transferred into the expression pTriEx2 (Novagen) vector, verified by DNA sequencing, and used to transform 2.2MALS experiments were carried out using an analytical Superdex S200 or S75 10/30 column  with online static light scattering , differential refractive index  and Agilent 1200 UV (Agilent Technologies) detectors.2.3DNA binding and its dependence on the probe length was simultaneously probed using molecular weight markers  while 500 nt long ssRNA transcript was used for RNA. Probes were mixed with increasing amounts of CHD4 constructs in 20 μl of binding buffer , incubated for 5 min at room temperature and then run on a 2% agarose gel. In the case of the titration of tPHD into tCHD, the proteins were mixed in 20 mM Tris pH8, 200 mM NaCl buffer and incubated at RT for 30 min.2.4X. laevis histones and a 168 bp fragment of 601-DNA (as described in Isolated radiolabeled mononucleosomes (50 fmol) reconstituted by salt gradient dialysis from recombinant 2.5Kd values were estimated from the concentration dependence of the steady-state response.SPR binding studies were performed using a Biacore 2000/3000 and T100  at 25 °C in 20 mM Tris pH 7.5, 200 mM NaCl, 0.05% (v/v) polysorbate 20. Biotinylated histone peptides (Millipore) were immobilized onto streptavidin sensor  2.62) at 30 °C as described in The steady-state rate of ATP hydrolysis was determined using the phosphate release assay EnzCheck (Invitrogen) in a standard buffer  in GNOM 3MWYThe solution small angle X-ray scattering (SAXS) data were collected using synchrotron radiation at ESRF beamline ID14-3 . Ten frames were collected and processed by in-house software. Background subtraction and data quality appraisal were done using PRIMUS program package 3In order to dissect the individual roles of the tPHD, tCHD and ATPase of the human CHD4 chromatin remodelling factor, we created a panel of domain constructs a. All th3.1We tested whether a minimal construct containing all three domains (tPHDtCHD/ATPase) of human CHD4 is able to bind dsDNA, whether the tCHD is responsible for such binding, and if this interaction is influenced by the adjacent tPHD and ATPase domains. The ability of different constructs to bind dsDNA was probed by mobility shift assays using a DNA ladder as a way of probing binding to differently sized molecules. The tPHDtCHD/ATPase retarded larger (>0.5 kbp) DNA fragments irrespective of ATP b. In conWe next tested the binding affinity of isolated tPHD and tCHD for dsDNA. When incubated with the tCHD, the dsDNA probe was retained in the loading well, forming complexes too large to enter the gel c unless . Interestingly, aggregation could be prevented by titrating tPHD into tCHD  exhibited dsDNA binding similar to that of tPHDtCHD/ATPase without aggregation c. In connto tCHD d which ynto tCHD . In conc3.2We tested the capability of the various isolated CHD4 domains to bind nucleosome core particles (NCPs). The tPHDtCHD/ATPase caused a significant shift in the mobility of the NCPs (50 fmol) , the magThese results demonstrate that the tPHD domain is necessary and sufficient for nucleosome binding. The tCHD and ATPase domains further increase the binding affinity presumably by binding to DNA that becomes transiently released from the core and/or by allosterically increasing the affinity for histone tails  or K9  (1–21 (Kd = 190 ± 14 μM) or to H31–21K9me3 (Kd = 70 ± 5 μM) but not to H31–21 methylated on K4 . The construct with all three domains, tPHDtCHD/ATPase, exhibited the highest affinity for H31–21 (Kd = 4.1 ± 2 μM), mostly due to a slower dissociation rate. Similar affinities were obtained for the binding to H31–21K9me3 (dK = 1.4 ± 1.0 μM) and ATP-γ-S while ADP was not effective (data not shown).Binding affinities of various constructs for histone H3 peptides were determined by surface plasmon resonance (SPR). The isolated tCHD showed no detectable binding to H32 and 3) . For theed on K4 , Fig. 3.–21K9me3 . Only a Together, our results indicate that the coupled tPHD and tCHD of CHD4 form a histone interacting moiety, whose binding affinity is augmented by inter-domain interactions with the ATPase domain through which ATP binding and hydrolysis may further modulate the binding.3.4kcat = 0.009 ± 0.0036 and 0.012 ± 0.0003 s−1, respectively) as shown in kcat increase at saturating DNA concentration) whereas it had no effect on the activity of the ATPase domain alone. This stimulation was specific for dsDNA since no effect was observed in the presence of RNA (polyA). Interestingly, and in contrast with what was shown previously for dMi-2 kcat = 0.001 ± 0.0002 s−1), which was not rescued by the addition of DNA and is close to the background. These results suggest that tPHD confers DNA-stimulated ATPase activity by modulating the way in which tCHD binds dsDNA and possibly by interaction with the ATPase domain. The tCHD on its own is not sufficient to confer DNA-stimulated ATPase activity, or perhaps as in yeast CHD1, it may inhibit the ATPase motor kcat = 0.0615 ± 0.0035 s−1), comparable to previously reported values Full-length recombinant CHD4 was shown to possess ATPase activity which is stimulated by nucleosomes and, to a lesser extent, by naked DNA kcat values are much lower than those observed for SWI/SNF, ISWI and CHD1. As previously shown the ATPase activity of the full length CHD4 is very low and about one-third that of the native NuRD complex It is interesting to note that tPHDtCHD/ATPase 3.52L5U and 2L752H1E3MWYThe overall shape and domain arrangement of CHD4 were studied by solution small angle X-ray scattering (SAXS). Scattering curves were checked for concentration dependence and radiation damage and aggregation and only data for monodisperse solutions were included in analysis. Guinier plot parameters were compatible with MALS and DLS analysis  indicatiDomain architecture of tPHDtCHD/ATPase e was obttPHDtCHD/ATPase structure can be divided into two parts: the head and the stalk. The seahorse-shaped tPHD contributes one arm to the stalk domain while the other arm contacts both the tCHD and ATPase within the head domain. tCHD extends towards the stalk contacting tPHD, but shares an extensive interface with the ATPase in the head domain. In effect, the ATPase, which constitutes the bulk of the head domain, is wedged between the arms of both CHD and PHD domains. The intimate association of all domains is fully consistent with the functional interdependence demonstrated above.4Chromatin remodelling ATPases play essential roles in organizing the chromatin landscape which regulates eukaryotic gene expression, yet their mechanism of action is poorly understood. ATP-dependent nucleosome remodelling is likely to be a multi-step process that involves binding of the remodeller to the nucleosome and ATP substrates, ATP hydrolysis, movement of the histone octamer relative to DNA and dissociation of the enzyme from the nucleosome. A tight coordination between these activities is required to achieve efficient remodelling.The simultaneous presence of tPHD and tCHD is a unique characteristic of the ATP-dependent chromatin remodellers CHD3, CHD4 and CHD5 . Perhaps, given the physical interaction between tPHD and tCHD domains in the stalk region, tPHD-mediated modulation of tCHD–DNA interaction could be explained by tPHD masking one of the DNA binding sites. Sequence analysis of tPHD shows that a long linker (32 a.a.), which is rich in acidic residues (15 a.a), connects PHD1 to PHD2, making it a plausible electrostatic mask that repels DNA from one of the binding sites on tCHD (In the present work we show that the two tandem domains of CHD4, tPHD and tCHD, are structurally coupled and modulate each other’s affinity for their respective substrates a. The tC on tCHD .Kd = 18 μM Kd obtained here for the tPHDtCHD (Kd = 14–19 μM, 1–12 with Kd values ranging from 3.2 ± 0.6 μM to 17 ± 5 μM The tPHD recognizes both un-methylated as well as K9-methylated N-terminal tails of histone H3. The affinity for the H3 tail is increased by the presence of tCHD which does not bind histones. Thus, the effect is most likely structural. The proposed intimate association between tCHD and tPHD domains within the stalk suggests that tCHD may stabilize an active conformation of tPHD and enable it to bind H3 with high affinity b. IntereIn our study, we show that the ATPase domain enhances the histone-tail binding affinity of the tPHDtCHD moiety. The ATPase domain greatly increases affinities compared to those observed for the isolated tPHDtCHD , Fig. 3.Vice versa, the tPHDtCHD moiety is essential for the DNA-stimulated ATPase activity since only the tPHDtCHD/ATPase shows DNA-stimulated ATPase activity . This reThus, the intertwined arrangement of DNA binding domains, histone tail binding domains and the motor domain in CHD4 might enable coordination of nucleosome binding and release with the ATP-dependent nucleosome remodelling activity b. In theThese results suggest that the conserved domains flanking the ATPase motor do not only direct CHD4 to the correct substrate but also participate in the remodelling activity. Since this combination of a motor domain and “chromatin targeting” domains is the unifying feature of all chromatin remodellers, we propose that inter-domain allosteric regulation might be a general feature of this class of enzymes.NOTE ADDED IN PROOF: Since this work was being revised a paper which describes a similar overall shape and domain interactions of CHD4 has been published


N. meningitidis PilQ: the N-terminus is shown to consist of two β-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/β fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/β domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/β domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the β-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from  Many bacteria which cause infectious disease in humans use large fibers, called pili, to attach to the surfaces of the cells of the host. Pili are also involved in a particular type of movement of bacteria, termed twitching motility, and the uptake of DNA into the bacterial cell. They are made up of thousands of copies of a specific pilin protein. The process of assembly of pili is complicated: it requires the cooperative action of a group of proteins which span both the inner and outer membranes in bacteria. Here we have determined the structure of part of the machinery which forms a channel between both membranes. One of the proteins, PilQ, is organized in a segmental way, being divided into separate domains which are jointed, hence allowing them to move relative to each other. We infer that this movement is critical to the functioning of the channel, which must open up to allow passage of the pilus fiber. We suggest that the function of the other protein we have studied, PilP, is to maintain the PilQ assembly during pilus secretion. Neisseria meningitidis, Pseudomonas aeruginosa and Vibrio cholerae−1) across surfaces N. meningitidis), a small protein which adopts an α/β fold and assembles into a helical structure which confers mechanical strength on the assembly Type IV pili are long (1–5 µm), mechanically strong polymers which extend from the surfaces of many Gram-negative bacteria, including Neisseria meningitidis showed a dodecameric structure, with a chamber sealed at both ends V. cholerae toxin, revealed a cylindrical-shaped structure with 12-fold symmetry enclosing a large chamber which is open at the periplasmic end but closed at the OM Salmonella needle complex: this shows the secretin in an open state, with the needle passing through both ends of the chamber The secretins are a large and diverse family of integral outer membrane (OM) proteins which comprise key components of the type II and type III secretion systems, as well as the biogenesis systems for type IV pili and filamentous bacteriophage N. meningitidis PilQ and two prototypical T2SS and T3SS secretins. All share a well conserved C-terminal region which spans the membrane and is responsible for oligomerization Neisseria meningitidisPseudomonas has shown that PilP also binds to PilO and PilN, two integral inner membrane proteins which are essential for pilus formation A number of proteins are known to interact with secretins, either for the purposes of assembly, OM insertion or mediation of function once the mature protein has been formed. Pilotin proteins are responsible for membrane targeting of secretins: the interaction sites of some have been mapped to the extreme secretin C-terminus, and their recognition of T2SS and T3SS secretins has recently been revealed at the structural level Here we report the structural determination of the PilQ periplasmic domains by using a combination of NMR and homology modelling. The original reconstruction of the PilQ oligomer which we reported was generated using cryonegative stain N. meningitidis, P. aeruginosa, Aeromonas hydrophila, Xanthomonas campestris and Xylella fastidiosa. We generally found the B2 domain more amenable to over-production and purification than B1 . The solution structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold . It is noteworthy that residues which are highly conserved in the B2 domain , which lie between the B1 and B2 domains and have been shown to influence the efficiency of TFP formation In contrast to the B2 domain, attempts to over-produce the B1 domains from several sources generally met with limited success: protein products were either produced in low yield and/or exhibited poor stability. The best progress was made with the B1 domain from -strands . The poo2 domain  are not N. meningitidis, N0N1PilQ343–545 (343–442) and its NMR structure determined by conventional methods using NOE restraints. The high quality structure adopts a fold similar to the N0 domains identified from GspD and EscC Secondary structure predictions and sequence alignments suggested the existence of two domains which are likely to adopt a variant of the α/β-type fold identified in other secretins Q343–545 , exhibitQ343–545 . Both th15N-1H residual dipolar couplings (RDCs) indicated that the N0 and N1 domains have no fixed orientation relative to each other in solution: it was therefore not possible to obtain a common orientation in the alignment tensor frame for the N0 and N1 domains from the RDC measurements. However, the rotation correlation times, calculated from the 15N T1 and T2 values obtained separately for the single N0 domain (τc∼9.6 ns) and the N0/N1 double domain (τc∼14 ns), suggest that the N0 and N1 domains do not tumble completely independently. It is likely that the helical part of the linker between the two domains reduces the flexibility in this region. We therefore generated 100 structures of the N0/N1 double domain using CS-ROSETTA Analysis of the 77–164 is a recombinant fragment which corresponds to the C-terminal domain of the PilP lipoprotein .PilPoprotein . Titratitructure . The revlP77–164 . The exp of PilQ . Similar77–164:N0N1PilQ343–545 complex. The largest HADDOCK-generated cluster bore marked similarities to the GspC-GspD complex 343–442 and PilP77–164 (PDB 2IVW), together with five intermolecular edge-on backbone hydrogen bond restraints (derived from the favored HADDOCK structure and related GspC-GspD complex), were input into CNS1.2 343–545 structure using CONSURF N. meningitidis PilQ in a similar manner to that for GspC and its cognate GspD secretin. This is therefore a further example of the congruence between the type II secretion and type IV pilus biogenesis systems.The identified residues involved in binding on the surface of each protein were used as input into the restraint-driven docking programme HADDOCK N. meningitidis PilQ oligomer, using negative stain-based methods We have previously reported on the structure of the intact Salmonellain vitroAlignment of the PilQ density map with the T2SS secretin VcGspD 224–329) and N0/N1 double domain (N0N1PilQ343–545) were docked into the cryoelectron density map using MULTIFIT, a program which has been shown to work well for structures with multiple components, even with low resolution maps 224–329 and N0N1PilQ343–545 which satisfied all the criteria . Why is it the case that expression of PilP is critical to TFP assembly in N. meningitidisThere are a number of well documented similarities between the proteins involved in TFP biogenesis and the T2SS: these include not just the secretins and cytoplasmic ATPases, but also structural components such as the cytoplasmic protein PilM, which has a similar fold to the T2SS protein EpsL The large periplasmic chamber formed by PilQ is reminiscent of similar structures found in other OM protein secretory complexes. The Wza translocon for capsular polysaccharides, for example, forms a more elongated chamber but it is also sealed at the periplasmic end Protocols for expression and purification of all proteins used in this study are described in 15N and 99% 13C labelled protein (1 mM-250 µM) in 500 µl of 90% 1H2O 10% 2H2O (or 100% 2H2O for specific experiments on N0N1PilQ343–545) in a solution containing 50 mM NaCl and 50 mM sodium phosphate at pH 6.8. All NMR experiments were carried out at 298K on Bruker Avance III 600 MHz and 800 MHz spectrometers equipped with TCI triple resonance cryoprobes. Spectra were processed using Topspin2.1 (Bruker) and the Azara processing package provided as part of the CCPN suite with assignment carried out using CCPN Analysis 1H13C and 1H15N HSQCs in conjunction 3D HNCA, HN(CA)CB, HN(CO)CA, HNCO, CBCA(CO)NH, HBHANH, HBHA(CO)NH, CB(CGCD)HD and HCCH-TOCSY experiments. Distance restraints were obtained from 3-dimensional 1H 1H 13C NOESY and 1H 1H 15N NOESYs using a mixing time of 100 ms. In addition, a set of experiments were collected on N0N1PilQ343–545 in 100% 2H2O; 1H13C HSQC, 1H15N HSQC, HCCH-TOCSY and 1H 1H 13C NOESY. Chemical shifts were referenced to DSS . Assigned chemical shifts of the first β-domain (B1) from Aeromonas hydrophilia, B2PilQ224–329, N0PilQ343–442 and N0N1PilQ343–545 were deposited in the BioMagResBank and assigned accession numbers 18419, 18459 and 18428 respectively. The Chemical Shift Index PilP and PilQ samples prepared for NMR analysis consisted of natural isotopic abundance or 98% 224–329 and N0PilQ343–442 were performed using CYANA 2.1 software 15N- and 13C-HSQC spectra. For B2PilQ224–329 a total of 4260 unassigned NOESY peaks were picked of which 3881 were assigned. For N0PilQ343–442 a total of 2677 unassigned NOESY peaks were picked of which 2337 were assigned. CYANA 2.1 calculations were ran with standard protocols using 7 cycles of automated NOE assignment and structural calculations, producing 100 structures per cycle. Structures were subsequently water-refined using CNS1.2 224–329 and a total of 1309 unambiguous interproton distance restraints and 110 dihedral restraints for N0PilQ343–442. Dihedral φ and ψ torsion angles were produced by TALOS+ http://nmr.cmbi.ru.nl/icing/iCing.html). Atomic coordinates and NMR restraints of B2PilQ224–329 and N0PilQ343–442 have been deposited in the Protein Data Bank under the accession codes 4AQZ and 4AR0 respectively. The secondary structure of B2PilQ224–329 was calculated using the STRIDE webserver Automated NOESY assignment and preliminary structure calculations of B2PilQ419–514) could not be determined using conventional methods due to the low number of unambiguous NOEs. Therefore 'C, Ha, Ca, Cb, N, and HN assigned chemical shifts were submitted to the CS-ROSETTA webserver 419–514 was identified using RCI 343–442 was also determined using CS-ROSETTA and compared with the NOE-derived structure. The helical linker region was included for both the N0PilQ343–442 and N1PilQ419–514 ROSETTA structure determination; in both cases the linker region consisted of a four turn helix comprising P419-E432.The structure of the N1 domain  again identified a set of peaks that attenuated upon binding. Spectra were collected at titration points of [PilQ]:[PilP]; 1∶0.1, 1∶0.2, 1∶0.3, 1∶0.5 and 1∶1. All spectra were collected and processed using TOPSPIN 2.1 .For the PilP- PilQ titration, a low concentration (50 µM) sample of 77–1641H 15N HSQC was assigned by transfer from BMRB star file 7209 PilPHigh Ambiguity Driven biomolecular DOCKing) 343–442 and the C-domain of PilP (PDB 2IVW) PilP100N-O351PilQ, PilP102N-O349PilQ, PilP102O-N349PilQ, PilP 100O-N351PilQ, PilP98O-N353PilQ. Using CNS1.2 343–442 and PilP (PDB 2IVW) structures. An ensemble comprising the 20 lowest energy structures was obtained. The advantage of this dual HADDOCK-CNS approach was that it overcame problems associated with rigid-body docking.Initial protein-protein docking utilized the restraints-driven docking program HADDOCK  from the digital micrographs using the EMAN software package startcsymm). Based on prior work et al.−1. Application of the more recently introduced, and more conservative, rmeasure software −1 resolution. Maps derived by electron microscopy were displayed with the CHIMERA software package 3 µl samples were applied undiluted to Quantifoil R 1.3/2 holey carbon-coated EM grids and blotted using Whatman No.1 filter paper (2×1 sec blots) at 90% humidity and then frozen in liquid ethane using a Vitrobot plunge freezing system . Cryo-EM was performed using a Tecnai F20 200 kV EM operating in low dose mode at 200 kV. Micrographs were recorded using a Gatan 4 k×4 k CCD at underfocus in the range 1–5 µm and with a calibrated magnification corresponding to 4.53 Å/pixel at the specimen level. Images were recorded under low-dose mode with an overall electron dose of 20- 25 electrons/ÅFigure S1Comparison of the folds of the B2 domain from N. meningitidis224–329) with the CS domain from human Sgt1. PilQ .(TIF)Click here for additional data file.Figure S2Structure-based sequence alignment of B2 domains. The locations of β-strands in the N. meningitidis structure are shown. Numbering is for the N. meningitidis sequence. Residues which are well conserved are highlighted in bold. Example sequences shown are Pseudomonas aeruginosa (Pa), Xanthomonas campestris (Xc), Aeromonas hydrophila (Ah), Legionella pneumophila (Lp) and Xylella fastidiosa (Xf) .(TIF)Click here for additional data file.Figure S3Chemical shift and deduced secondary structure assignments for the B1 domain from Aeromonas hydrophila. Top: CSI calculated for deviations from random coil shifts of Hα, Cα and CO to determine the consensus secondary structure, graph adapted from CCPN analysis. Bottom: alignment of B1 domain in A. hydrophila (Ah), N. meningitidis (Nm), P. aeruginosa (Pa) and L. pneumophila (Lp) . Residue sequence and numbering for the A. hydrophilia sequence incorporates the loss of the signal sequence and residues from the expression vector.(TIF)Click here for additional data file.Figure S4343–545 and N0PilQ343–442 from Chemical shift and deduced secondary structure assignments for N0N1PilQNeisseria meningitidis. CSI calculated for deviations from random coil shifts of Hα, Cα and CO to determine the consensus secondary structure; graph adapted from CCPN analysis.(TIF)Click here for additional data file.Figure S577–164.Ratio of PilQ:PilP at the point of PilQ NH peak attenuation in the PilQ N0 and N1 domains on binding of PilP Binding of PilP77–164 to N0N1PilQ343–545 is shown in blue and N0PilQ343–442 in red.(TIF)Click here for additional data file.Text S1Protein expression and purification. Methods for the expression and purification of all proteins used in this study.(PDF)Click here for additional data file.


Lβ2) and ICAM-1 derive solely from the integrin αL I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the αL I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine αL I domain allostery between the C-terminal α7-helix  and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the αL I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to αL I domain but reduced interaction with HRV.Intermolecular contacts between integrin LFA-1 (α Integrins are noncovalently associated αβ heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix adhesions, signaling bidirectionally across the plasma membrane. Integrins play important roles in development, immune cell trafficking and responses, and homeostasis i.e. allostery in α I domains, has been demonstrated by rationally designed mutational studies Previous structural studies have indicated that the I domains of both α and β chains exhibit low to high affinities to their ligands The ectodomain of ICAM-1 contains five Ig-like domains with the first domain D1 solely responsible for interactions with LFA-1 and HRV. The binding sites in D1 for LFA-1 and HRV, however, are distinct. The loops at the N-terminal face of D1 interact with HRV by docking into a region known as the canyon M and α2 I domains Here we report the crystal structure of the complex between the engineered LFA-1 I domain and ICAM-1 D1. Distinct from the previous studies L I domain, which maintains a modular function in isolation, has been engineered to high affinity by mutations, such as a pair of cysteines (K287C/K294C) to lock the α7-helix in an active conformation 2+) of the MIDAS , notably a total of 14 complexes per asymmetric unit with a large unit cell dimension were found in our structure . This ma its own .The LFA-1 I domain has previously been cocrystallized with ICAM-1 D1D2 Unlike the retention of a modular function of the I domain on its own, functional expression of D1 by itself was achieved only after the introduction of seven mutations  into Gln1-Thr85 sequence, identified by the combination of directed evolution and rational design approaches i.e., T23A and S67A) in turn would have affected the interaction with the neighboring residues, thus placing the B–C loop closer toward the protein core  or in D1D2 in complex with the LFA-1 I domain (0.6 Å RMSD to 1MQ8) . The larein core . In contein core  & Fig. 4Here we report the crystal structure of the complex of LFA-1 α I domain and ICAM-1 D1, which have been engineered for high affinity and native fold, respectively. A point mutation in the LFA-1 I domain (F265S), which resulted in an increase in affinity to ICAM-1 by ∼10,000 fold over the wild-type I domain M I domain structure with glutamic acid of the neighboring I domain coordinating to the metal ion in the MIDAS 2 I domain bound to a collagen mimetic peptide, exhibiting a similar downward position for the α7-helix The structural basis of allosteric switching of the integrin I domains to the conformation that is competent for ligand binding has been studied extensively. Conformational allostery in the α7-helix was first evidenced in the αi.e., the importance of hydrogen bond network in the protein core that determines the conformation of the CDR-like loops for interaction with HRV, may provide a further insight into designing a functional D1 alone that retains full capacity binding to the virus. Functional D1, due to its low-cost production from bacteria, may be developed into a decoy antagonist to block LFA-1/ICAM-1 interactions and HRV binding to ICAM-1 expressing host cells.ICAM-1 consists of five Ig-like domains in its extracellular region, of which D1 is solely responsible for the molecular contacts with the LFA-1 I domain and HRV. Despite an extensive set of mutations in D1, which would convert mainly hydrogen bond interactions into vdW contacts, the D1 mutant was found to retain a native conformation of β-strands and β-sandwich fold with a largest deviation localized to the loops that form the N-terminal face of the Ig-like fold. Conformational variability of the loops in D1, analogous to the complementarity determining region (CDR) in antibodies 2 to form the complex, purified by size exclusion and ion exchange columns, and concentrated to 11 mg/ml in 50 mM HEPES, pH 7.2, 10% glycerol. Protein complex was then mixed at equal volume with the buffer for crystallization . Crystals were grown in a sitting drop at room temperature.The LFA-1 I domain (Asn-129 - Tyr-307 with a mutation F265S) and ICAM-1 D1  were expressed in E. coli BL21 (DE3) (Novagen), refolded, and purified as previously described L I domain (PDB codes 1MQ8 and 3BN3) as search models. Then the structures were refined with Refmac5 L I domains . Considering relatively low diffraction resolution (3.6 Å), tight non-crystallographic symmetry (NCS) restraints were applied to the 14 complexes. The refined structures were proofread and corrected with COOT free factors converge to 21.8% and 23.4%, respectively. The structural refinement using PHENIX free values. The final structures were validated with PDB validation server (www.pdb.org). The coordinates of our complex structures have been deposited to the RCSB with the PDB code 3TCX. Details in data collection and structure refinement can be found in The diffraction data were collected at Cornell High Energy Synchrotron Source (CHESS) Beamline F1 and processed with HKL2000 M (1IDO) 2 (1DZI) L (3F74) L I domain of our structure (3TCX) based on residues in the α6-helix   L I domain of our structure (3TCX) using residues in the central β-sheet . Previous wild-type ICAM-1 D1 structures (1IAM Previous structures of the α268–278) . Previoures 1IAM . All the