Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals.G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan  The discovery of activation-dependent rhodopsin phosphorylation almost 40 years ago Regulator of G protein Signaling (RGS) homology domain (RH) Based on the relative extent of sequence similarity, vertebrate GRKs have been traditionally divided into three subfamilies: GRK1, comprising GRK1 (rhodopsin kinase) and GRK7 (cone opsin kinase); GRK2, comprising GRK2 and 3; and GRK4 comprising GRK4, 5, and 6. All GRKs are multidomain proteins  GRK (gi 4519169) was performed, as described in B. floridae and C. intestinalis, fruit fly and other insects, nematode C. elegans, sea anemone N. vectensis, and primitive metazoan T. adherens. Interestingly, we also detected the RH+KD fusions in non-metazoan species, including the opisthokont Monosiga brevicolis, thought to be the unicellular eukaryote closest to Metazoa Capsaspora owczarzaki (ATCC 30864), a unicellular amoeboid parasite of tropical snails, that has recently emerged as another candidate sister clade of Metazoa Phytophotra infestans; white rust oomycete Albugo laibachii Nc14; and brown alga Ectocarpus siliculosus. All sequenced genomes of invertebrates and of a cephalochordate have two GRK-like genes, whereas all unicellular organisms have only one.To extract GRK sequences, the psi-blast search with the kinase domain of We have extended these observations by building phylogenetic tree  based onC. elegans, there are two GRK genes, one of which, called grk-2, encodes a product with 66% identity with human GRK3 and similar domain structure Drosophila, one of the two GRK isoforms, photoreceptor-enriched GRK1, is highly similar to the mammalian GRK2 and GRK3 and has the same domain structure B. floridae) GRKb, to avoid the impression of their specific association with mammalian GRK1 branch . Interestingly, the sole known GRK-like protein in unicellular Monosiga is placed into this group on the basis of similarity in KD with moderately significant bootstrap support of 65%, and it also has the C-terminal PH domain.All proteins in the GRK2/3 clade, from primitive metazoans to vertebrates, have an additional shared feature, the C-terminal PH domain. In C. elegans, this clade is represented by grk-1 gene product and in Drosophila by ubiquitous GRK2; in B. floridae. The sole known GRK homolog from opisthokont C. owczarzaki is also robustly placed into this clade (bootstrap 92%).The other clade is split into two subgroups, vertebrate-specific clade consisting of rhodopsin (GRK1) and cone opsin (GRK7) kinases, and the other clade comprising mammalian GRK4, 5 and 6 and their single-copy orthologs in lancelet and invertebrates. These proteins are more diverse than the members of the GRK2/3 clade. In C. intestinalis appears to represent an intermediate step in the evolution of the two clades. It has three GRKs, one of which is a basal member of GRK4/5/6 clade (renamed GRKa for this reason), another (GRK1) is a basal member of the “visual” branch in the same clade  ortolog into a separate branch (bootstrap 85%). It also lacks any recognizable C-terminal motifs typical of the GRKa/4/5/6 family, although the pufferfish ortholog has a characteristic amphipathic helix. In the zebrafish genome, a pair of sequences has been annotated as GRK1 (designated GRK1a and GRK1b) and GRK7 (GRK7a and b)  .Fourth, in addition to the three main branches in the tree, the GRK-like putative proteins from oomycetes and an alga form their own clade, apparently preceding the split into GRKa/1/7/4/5/6 and GRKb/2/3 in Metazoa and their unicellular relatives.The ancient origin of the characteristic RH+KD domain organization of GRKs is also supported by the analysis of the intron positions in the genes of GRK2/3 clade, the vast majority of which are found in the orthologous codons in higher and lower metazoans, suggesting the shared exon-intron structure of the ancestral GRK2/3 gene . The exoN.vectensis, placozoan T. adherens, and non-metazoan M. brevicolis. Multiple residues and the overall helical structure are conserved in GRKs from monocellular oomycetes Phytophtora, Albugo, and brown alga Ectocarpus, supporting tentative placement of predicted GRKs from these organisms into GRKb/2/3 clade based on the kinase domain alignment and phylogenetic tree analysis  and GXS core, and two out of three typical basic residues are substituted by non-charged amino acids.The N-terminal sequence of most Metazoan GRKs in the GRKa/4/5/6 clade contains another characteristic element. Predicted α-helix is followed by several structure-breaking glycines, followed by XXarginine . In the GRKs are soluble, whereas their best-characterized substrates, GPCRs, are integral membrane proteins. GRK membrane localization is mediated largely, although not exclusively, by special sequence motifs in the C-termini, and GRK proteins have evolved different mechanisms for recruitment or attachment to the plasma membrane [reviewed in D. rerio) that did not have any recognizable C-terminal motif. C. elegans and C. intestinalis have two characteristic pairs of hydrophobic residues (FFXXLF motive found in GRK4) surrounded by many conserved positively charged residues. In B. floridae, leucine in the second pair is substituted by threonine, but there are two putative palmitoylation residues as in GRK6A, which are absent in C. elegans or C. intestinalis. The insect species evolved a unique C-terminal sequence consisting of a hydrophobic pair CF (not found in other species except cnidarian N. vectensis) followed by PF and a stretch of positively charged residues. Although three hydrophobic residues are present, the amphipathic helix is less likely due to the presence of three prolines; overabundance of positively charged residues suggests that in insect GRKs this element is more likely to be a membrane-binding positive patch  as well as positively charges residues, which may be due to a secondary loss. The GRK of the opisthokont C. owczarzaki, the earliest known GRK of the GRK4/5/6 subfamily, is missing phenylalanine in the second hydrophobic pair. However, close by there are two more hydrophobic residues, leucine and phenylalanine, able to form a stretch of hydrophobic residues on one side of the helix . That includes core residues , D85, and E87 (the latter is the first amino acid in the α-helix) are highly conserved. In GRK from B. floridae all three residues are present, whereas in C. intestinalis D85 is substituted by glutamine. Invertebrates are missing this particular triplet, but they have their own arrangement of negatively charged amino acids consisting of D84-E85 pair. Cnidarian H. magnipapillata retains the same arrangement as in vertebrates, D83-D85-E87, whereas T. adhaerens and M. brevicolis only have E83 and no other charges.An important feature of PH domain is a conserved distribution of charged residues forming positively charged surface on one side and negatively charged on the other his loop . Anotherbona fide PH domain  normally found in cone kinases (GRK7s) that would allow these GRK to semi-permanently associate with the plasma membrane.The earliest GRKs from oomycetes and an alga, which form their own clade loosely associated with the GRKb/2/3 subfamily on the basis of aligned KD domains, lack recognizable lipid-binding motifs found in the GRKa/4/5/6 subfamily, nor do they possess a H domain . Howeverwww.kinase.com), which is ubiquitous in unicellular eukaryotes. Thus, it is most likely that the KD region of GRKs has been produced by duplication of the S6 kinase in the evolution of protists. The provenance of the RH domain is much harder to establish because of the fast evolution of this relatively short region and the uncertain placement of the RH domain of GRKs in the phylogenetic tree of RGS proteins Future phylogenetics analysis, including more extensive taxonomic sampling of unicellular relatives of Metazoa, invertebrates and primitive vertebrates will provide a more detailed understanding of evolutionary events that shaped the GRK family, including the picture of gene duplications and losses and, perhaps, of unequal evolutionary rates within the family , first at the root of the vertebrate lineage and the second when jawless vertebrates brunched off , which has recently become available The GRKa clade later split into GRK1/7 and GRK4/5/6 lineages – most likely in the early chordates, since an apparently ancestral “visual” GRK is present in the urochordate A third full-genome duplication is believed to have occurred in teleost fish lineage Monosiga, have C-terminal PH domains; all GRK1/7 proteins have either farnesylation or geranylgeranylation CAAX motifs; and all GRK4/5/6 proteins have a predicted C-terminal amphipathic helix in one form or another. The shape of the helix with its characteristic arrangement of hydrophobic and positively charged residues specific for each GRK isoform is strictly conserved in vertebrates. In invertebrates lacking multiple GRK isoforms in the GRK4/5/6 clade, such as Chordata species lancelet and sea squirt, nematode C. elegans, placozoan T. adherens, and opisthokont C. owczarzaki, the form of the helix is GRK4-like, suggesting that this might be the ancestral form preceding gene multiplication in this clade. Insect species have their own unique strictly conserved arrangement, similar to that in cnidarian Nematostella vectensis. Thus, different arrangements of the lipid binding residues in the helix appeared in evolution of the GRKa/4/5/6, but some later disappeared or were modified. Although the precise mode of membrane interaction of invertebrate GRKa is unknown, distinct conservation of amphipathic helix suggests a mechanism similar to that operating in mammals.Split of the ancient eukaryotic GRK into opisthokont-specific GRKa and GRKb appears to have been followed very closely by acquisition of two distinct C-terminal extensions, a PH domain in GRKb and alternative membrane-targeting sequences in GRKa. The large partitions in the GRK kinase domain family trees show close correlation with the identity of the C-terminal regions of GRKs that are involved in membrane binding . All proGallus gallus rhodopsin kinase robustly placed in the GRK1 group has CGVL geranylgeranylation motif. The basic member of the “visual” group, a GRK from C. intestinalis, also has a geranylgeranylation motif CALL. Thus, geranylgeranylation may have been the lipid modification in an ancestral GRK in the “visual” branch that later changed to farnesylation in the GRK1 subfamily.The GRK1/7 clade is characterized by the presence of a C-terminal prenylation CAAX motif mediating membrane attachment of the members of this GRK subfamily Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin) GPCR families could be traced to common ancestor of Uniconts and Alveolates at the very root of eukaryote evolution M. brevicolus genome encodes an arrestin-like protein; C. owczarzaki genome encodes two, whereas other GRK-containing unicellular Chromalveolate eukaryotes do not appear to encode any. However, GRKs from unicellular oomycetes and brown algae, although quite different from metazoan GRKs, have perfectly recognizable N-terminal receptor-binding motive  was run with default parameters, except that more matches were allowed to be reported (-b and -v parameters both set to 5000). After two iterations, all known and predicted GRKs from the model organisms were extracted together with many proteins containing RH+KD fusion not annotated as GRKs, followed closely, and sometimes interspersed with, ribosomal protein S6 kinases (RPSK), at E-values less than e−10. After verifying that all sequences above this threshold were members of different kinase families, we selected the gi numbers of the top 386 sequences that included many RPSKs, extracted these sequences from the NR database and removed all RPSKs. RPSKs were excluded based on the absence of RH domain. Furthermore, RPSKs group together, at the exclusion of GRKs, in single-linkage clustering and in the phylogenetic tree when they are included in the phylogenetic analysis (data not shown). GRK sequences with incomplete KD, shorter alternatively spliced variants, and GRKs from partially sequenced or incompletely annotated genomes . The resulting sequences were aligned using the MUSCLE program The homologs of GRK proteins were collected with the aid of the BLAST family of programs Table S1GRK sequences used in this study.(DOC)Click here for additional data file.


Mucosa-associated lymphoid tissue 1 (MALT1) plays an important role in the adaptive immune program. During TCR- or BCR-induced NF-κB activation, MALT1 serves to mediate the activation of the IKK (IκB kinase) complex, which subsequently regulates the activation of NF-κB. Aggregation of MALT1 is important for E3 ligase activation and NF-κB signaling.2 and contains numerous hydrogen and salt bonds. In conjunction with a second interface, these interactions may represent the basis of MALT1 oligomerization.Unlike the isolated CARD or paracaspase domains, which behave as monomers, the tandem Ig-like domains of MALT1 exists as a mixture of dimer and tetramer in solution. High-resolution structures reveals a protein-protein interface that is stabilized by a buried surface area of 1256 ÅThe crystal structure of the tandem Ig-like domains reveals the oligomerization potential of MALT1 and a potential intermediate in the activation of the adaptive inflammatory pathway.enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in This article can also be viewed as an  Mucosa-associated lymphoid tissue (MALT) lymphoma is a low-grade tumor composed mainly of B-cells characterized by chronic inflammation The biological role and function of MALT1 is related to the adaptive immune response, playing an important role in signal transduction, specifically in antigen B-cell receptor activation Activation of many E3 ligases is associated to their oligomerization or aggregation state, but the precise mechanism of activation is unclear The CMB complex is held together by many points of interaction, including an interface between the MALT1 death and tandem Ig-like domains with Bcl10 The death domain (DD) superfamily comprises of the following subfamilies: the death domain (DD), the death effector domain (DED), the caspase recruitment domain (CARD), and the pyrin domain (PYD). CARD domains participate in the assembly of oligomeric signaling complexes by mediating homotypic interaction with other DD superfamily proteins Clustering mechanisms of signaling molecules are the basis of adaptive and innate immune signal transduction. And these mechanisms are likely the molecular basis of how cIAP2-MALT1 fusion proteins activate the inflammatory pathway without upstream activators. In order to better define the oligomerization potential of MALT1, we solved the structure of the MALT1 N-terminal CARD-like death domain and the tandem Ig-like domain. Here we show using structural biology that unlike the CARD-like death domain, the tandem Ig-like domains naturally form oligomers with a tendency towards dimers and tetramers. Structures reveal the molecular basis of dimerization and tetramerization by the Ig domain and suggest that MALT1 oligomerization may be mediated at least in part by its tandem Ig-like domains. This study may help understand how MALT1 acts as an oligomeric scaffold protein to bind co-factors like Bcl10, CARMA1, and TRAF6, and activate the NF-κB pathway.The human MALT1 N-terminal CARD domain (residues 29–126) was 1) cloned into a pET28 vector system, 2) overexpressed in transformed BL21 bacteria, 3) purified using IMAC and size-exclusion chromatography, and 4) crystallized in potassium tartrate. Its structure was determined using a selenomethionine derivative . It is csee subsequent sections). Taken together, this suggested that instead of homodimerization, the CARD domain might be competent to heterodimerize.The unusual position of the sixth helix is also found in the crystal structures of another CARD containing protein, NOD1, where it is involved in a domain-swapping mechanism. The sixth helix sits in its canonical position, between helices A and E, but in another molecule. This was suggested to mediate homodimerization of NOD1. Unfortunately, the presence of a disulfide bond between the molecules clouded the conclusions CARD-containing MALT1 binding partners include Bcl10 and Caspase 8 Figure S1). As with MALT1, the Bcl-10 CARD model adopted a polarized charged surface, with helices B, E and F being acidic, and helices A, C, and D (the opposite face) being basic. These complementary surface charges may mediate heterodimerization, but further verification undoubtedly would be required. Unfortunately, nothing could be gleaned from phylogenetic analyses, because although the amino acid sequence of the last three helices of the MALT1 CARD domain was conserved, almost all of the conserved residues occurred in the core of the domain; in other words, no large surface-exposed patch of the domain was conserved  with Z scores greater than 10, including Musk , Axonin1 , Roundabout1  and Titin  (data not shown). Variations in loop structures occured almost exclusively at the C–D and D–E loops (The second MALT1 Ig-like domain (IgL2) was crystallized by itself and as a tandem domain with IgL1 . No confThe two Ig domains were arranged in a head-to-tail fashion, nearly parallel to each other, with a stagger and overlap that brought β-strand A near β-strand B'  and the C-terminal residue of IgL2 (Glu310). The reason for keeping the CARD and catalytic domains apart from each other would be unclear.Figure S2). The plots of ln (abs) vs the radius square were not linear and showed an upward trend, confirming that the sample had a significant level of intermolecular interaction. The global self-association was fit and the deduced average molecular weight was 75,940 kDa. The ratio of the average observed MW to the calculated MW was 3.5: 1.To explore the possibility that the N-terminal domains of MALT1 may adopt oligomeric states that would contribute to E3 ligase activation, we examined their intermolecular interaction in solution. Pure CARD and tandem IgL domains were analyzed by size-exclusion chromatography. Unlike the separate CARD and IgL2 domains, which ran as monomers, the tandem IgL1–IgL2 domains with theoretical molecular weight 23 KDa, ran as a sharp peak with an average estimated molecular weight corresponding to a tetramer (around 101 KDa) . In this assembly, IgL2 made all interactions, the most notable of which was a joining of β-sheets through the a″ β-strands in an anti-parallel fashion . Together these observations support a biologically relevant role for these interactions.To examine how MALT1 might oligomerize, crystal packing and higher-order associations were examined using the PISA server  Interestingly, if both assemblies co-exist in the same complex, an interlaced doughnut-shaped tetramer is formed . The column was washed with 10 column volumes of wash buffer . Protein was eluted with 2 column volumes of elution buffer . Protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column . Fractions containing protein were concentrated to a final value of 10 mg/ml, and stored in 20 mM Tris, pH 8.0, 500 mM NaCl, 5 mM DTT.The coding region of MALT1 CARD (residue 29–124), tandem Ig domains (residue 127–310), and IgL2 domain (residue 225–309) were cloned using a Mammalian Gene Collection cDNA template (AU79A6) into the pET28a-LIC vector (GenBank) using the In-Fusion CF Dry-Down PCR Cloning Kit (Clontech). Competent BL21 (DE3) cells (Invitrogen) were transformed and grown using the LEX system (Harbinger BEC) at 37°C in 2 L bottles (VWR) containing 1800 ml of TB (Sigma) supplemented with 150 mM glycerol, 100 µM Kanamycin and 600 µl antifoam 204 (Sigma). At OD (600) = 6.0, the temperature was reduced to 15°C, and one hour later the culture was induced with 100 µM IPTG and incubated overnight (16 hours) at 15°C. Cell pellets were resuspended in 30 mL per liter bacterial culture of lysis buffer , cells were lysed by sonication  on ice for 10 minutes. After centrifugation at 40,0002, 0.1 M Bicine, pH 9.0. Suitable crystals were cryoprotected by immersion in well solution supplemented with 15% (v/v) glycerol prior to dunking and storage in liquid nitrogen.Crystals of the N-terminal CARD domain were grown at 18°C using the hanging drop method by mixing equal volumes of protein (10 mg/ml) and crystallization buffer containing 1.0 M K/Na tartrate, 0.1 M MES pH 6.0. The second Ig domain crystals were grown in 30% PEG 550-MME, 0.2 M Ammonium sulphate, and 0.1 M Sodium cacodylate at pH 6.5 in hanging drops. Crystals of the tandem Ig domains were grown at 18°C using the sitting drop method by mixing equal volumes of protein (10 mg/ml) and crystallization buffer containing 1.5 M MgClDiffraction data of the CARD domain, IgL2 and tandem Ig-like domains of MALT1 were collected at beamline F1 at the Argonne Photon Source, Beamline 19-ID; beamline F1 at the Cornell High Energy Synchrotron Source; beamline CMCF-1 at the Canadian Light Source, respectively. The data was processed with HKL-2000 Sedimentation equilibrium was carried out using a Beckman Optima XL-A analytical ultracentrifuge with an An-60 Ti rotor. Tandem IgL domains in 20 mM Tris-HCl pH 8, 200 mM NaCl and 1 mM TCEP was spun at 12,000 and 15,000 rpm at 4°C. Absorbance was recorded at 230 and 280 nm. Three sample concentrations were used for each analysis: 0.25, 0.5 and 1.0 mg/ml. Data analysis was performed using the Origin MicroCal XL-A/CL-I Data Analysis Software Package Version 4.0.MALT1 domains at 10 mg/mL concentration in 50 mM Tris buffer, pH 8.0, 500 mM NaCl, 1 mM tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) were loaded onto superdex-200 16/60  size exclusion column at 4°C. Standard proteins included thyroglobulin (670 kDa), globulin (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), anhydrase (29 kDa), myoglobin (17 kDa), and ribonuclease (13.7 kDa).Figure S1Electrostatic CARD surface. Surface charge features of CARD domain of MALT1 (upper) and Bcl-10 model (lower). Two orientations are shown.(TIF)Click here for additional data file.Figure S2Analytic ultracentrifugation. Sedimentation equilibrium centrifugation analysis of the MALT1 tandem IgL1–IgL2 domains. The absorbance was measured at 280 nm at speeds of 12,000 rpm at 4°C. The lower panel shows the sedimentation equilibrium ultracentrifugation data for tandem IgL1–IgL2 domains at 1 mg/ml. Points shown were collected at 15,000 rpm and 4°C.(TIF)Click here for additional data file.Figure S3Electrostatic surface of dimeric MALT1 tandem Ig-like domains. (A) In the first dimer assembly of tandem Ig-like domains, two molecules were anti-parallel to each other in a cross-braced form. (B) The second dimer assembly of tandem Ig-like domains forms a tetramer. The electrostatic surface of the two tandem domains is presented in an open book style.(TIF)Click here for additional data file.Datapack S1http://www.molsoft.com/icm_browser.html.Standalone iSee datapack - contains the enhanced version of this article for use offline. This file can be opened using free software available for download at (ICB)Click here for additional data file.Text S1Instructions for installation and use of the required web plugin (to access the online enhanced version of this article).(PDF)Click here for additional data file.


Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles.The dicistronic  An integral membrane component of SV, SYT-1 is essential for fast Ca2+-dependent release of neurotransmitter in Drosophila, C. elegans, and mouse 2+-dependent complexes with the SNARE proteins and hence trigger exocytosis Drosophila have given insight into the essential function of the C2A and C2B domains of SYT-1 null flies, as indicated by the absence of fast neurotransmitter release 2+ cooperativity for synchronous neurotransmitter release and a defect in endocytosis as indicated by depletion of SV, which suggest that the C2B domain is essential for SV cycling. The AD1 phenotype is thought to be due to the inability of SYT-1 to bind the SNARE complex, and the inability of SYT-1 to oligomerize 2+ sensor for neurotransmitter release, SYT-1 has also been implicated in endocytosis of fused SV. The C2B domain of SYT-1 binds a number of important proteins and moieties associated with exo- and endocytosis, such as syntaxin, SNAP-25, μ2 and α-adaptin of AP-2, phospholipids and the Stoned proteins 2+-independent and via the lysine-rich motif 2+ and phosphatidylserine-dependent and to involve the µ2 subunit of AP-2 interacting with the lysine-rich motif of C2B SYT-1 Synaptotagmin-1 (SYT-1) is primarily associated with CaDrosophila, while this lysine-rich motif may be important for defining the size of recycled synaptic vesicles and in the priming/docking step of vesicle exocytosis 2+ channel and AP-2 In 2+-binding loops of the C2B domain, when present in transgenic Drosophila, results in the reduction of SV endocytosis to the equivalent of SYT-1 null mutant allele Two other regions of the C2B domain have been implicated in retrieval of SYT-1 from the plasma membrane. It has been shown that a tryptophan containing motif, present in the last β-strand of the C2B domain (WHTL), can act as an internalization signal in chimeric proteins expressed in PC12 cells 2+. The observed evoked transmitter release is decreased by 95% and the apparent Ca2+ affinity for synaptic transmission is also reduced null mutant allele. The slower rate of endocytosis in D3,4N flies, is different from that in SYT-1 null flies, as D3,4N flies can be rescued by increasing extracellular Ca2+, suggesting that Ca2+ coordination is important for regulating the rate of endocytosis Mutation of the C2B residues Asp416 and Asp418 to asparagines  caused a defect in exocytosis due to the disruption of bound Ca2+-triggered rearrangements between proteins and lipids are believed to cause the opening and dilation of a fusion pore which subsequently leads to the exocytic release of the vesicle contents. Ca2+ is postulated to first drive C2 domains membrane interactions, which result in a conformational change that triggers a subsequent C2B-mediated oligomerization step Drosophila, mutations at the stoned locus are known to affect the rates of SV endocytosis and alter the presynaptic distribution of SYT-1, even though the distribution of endocytic proteins such as dynamin, α-adaptin and clathrin, are unaltered 2+-mediated conformational change of SYT-1 may be important in mediating the interaction of the Stoned proteins with SYT-1.Much research has focused on the role of the AP-2 complex in SYT-1-induced SV endocytosis. Phospholipids have been shown to interact with SYT-1 Drosophila stoned locus is dicistronic, encoding two polypeptides, Stoned-A (STNA) and Stoned-B (STNB) Drosophila intersectin, DAP-160 The Drosophila STNA and STNB proteins have been primarily studied through their interactions with SYT-1 shibire, does not prevent STNB-bound vesicles from being recovered, from, presumably, inactive synapses in these dark adapted flies 2+ may play a role in this interaction. When Drosophila heads are homogenized in the presence of Ca2+, STNB and SYT-1 are found almost entirely in the plasma membrane fraction while in the absence of Ca2+, both STNB and SYT-1 are found in both plasma membrane and vesicle enriched fractions 2+ may have an important role in the binding of the Stoned proteins to SYT-1. Exactly what the role might be for the Stoned proteins in SV recycling might be, is unclear, yet they would appear to be essential for normal SV recycling in the fly. We therefore continue with our study of these proteins and especially with their interaction with SYT-I. Here we report our investigations of the effect of Ca2+ and phospholipids on the binding of the CCD of STNA and the µHD of STNB to the purified recombinant C2B domain of SYT-1. We have found that Ca2+ greatly increases the binding of STNA and STNB to the C2B domain of SYT-1, that these interactions are primarily driven by a Ca2+-dependent self-association of the C2B domains of SYT-1 molecules and that the likely binding site for STNB resides within the Ca2+-binding loops of the dimeric or multimeric C2B protein domains. We have also observed a negative effect of phospholipids on the in vitro binding of STNB to the C2B domain of SYT-I.The possible roles of the 2+ considerably enhanced the binding of both MBP-STNB (MHD) and MBP-STNA (CCD) proteins to the GST SYT-1 C2B domain. For STNA this increase was 2.5 fold  while for STNB it was 4 fold . The level of binding of the MBP-STNA and MBP-STNB proteins to GST-C2A was slightly higher than to GST alone. A report has shown that the C2A domain is also capable of binding Ca2+, even though, due to incomplete Ca2+ coordination, the C2A domain has a low intrinsic in vitro affinity for Ca2+2+-free buffer (approximately 30% of binding in Ca2+ buffer), and might suggest that the observed binding of STNB and SYT-I in the presence of EGTA represents non-specific binding and that Ca2+ plays an essential role in the binding of the Stoned proteins to C2B SYT-1. We had previously shown that STNA and STNB both bound preferentially to the C2B rather than the C2A domain of SYT-1 2+, both STNB and STNA can be dissociated from the C2B domain if the complexes are subsequently washed in EGTA-containing buffer .After being bound in the presence of Cain vivo2+ and phosphatidyl serine (PS) 2+-dependent binding of MBP-STNB to GST-C2B is completely inhibited by the presence of Triton X-100-produced micelles containing phsopholipid mixtures of phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and phosphatidyl serine (PS) as compared with the binding in the presence of Ca2+ alone and that the critical phospholipid appears to be PS  alters the rate of exocytosis 2+ concentration to undergo exocytosis 2+ dependent oligomerization renders C2B SYT-1 inefficient for exocytosis. It was reported that Ca2+-triggered SYT-1 clustering is via the C2B domain and is required for exocytosis 2+-dependent endocytic complex. However, a previous study has shown that STNB can be found bound to synaptic vesicles, via SYT-1, prior to exocytosis 2+ influx that might trigger SYT-1 oligomerization and the coupling of excitation and vesicle fusion 2+, or that other factors, such as those that might alter the structure of the C-terminal region of SYT-I C2B, are playing a role in potentiating the binding of STNB to SYT-1 even in the absence of Ca2+. Certainly there is no STNB protein in the soluble fraction from fly head extracts Upon CaDrosophila, but also exocytosis defect by decrease evoked transmitter release and reduce in apparent Ca2+ affinity for synaptic transmission stoned mutants showed alterations in both spontaneous and evoked release at larval NMJ and severe neurotransmission defect that may lead to embryonic lethality rd loop of C2B SYT-1 can outcompete the µ-homology domain of STNB (our data), suggesting the interaction of STNB to SYT-1 is mediated by a region in loop 3 of C2B domain. Thus, it is an attractive hypothesis that STNB may act as an inhibitor for membrane fusion. In a recent paper 2+; while the binding of PIP2 with SYT-1 is less affected by Ca2+. It would be interesting to investigate whether these lipids will have similar effect as PS in affecting Stoned and C2B SYT-1 binding.The D3,4N mutation in transgenic flies results in a reduction in the rate of endocytosis of synaptic vesicles in vivo conditions as STNB. The presence of putative AP-2 binding motifs in STNA 2+ has a marked effect on STNA and STNB association with SYT-1 and confirms them as important proteins in the mechanism(s) of synaptic vesicle recycling in Drosophila.The role of STNA in the synaptic vesicle cycle remains elusive. The presence of STNA at the larval NMJ appears nonessential E. coli. The C2A construct (bases 137-326) was produced as described previously Drosophila syt cDNA clone using a 5′ primer that generated an EcoR1 site (5′CTGGTCAGCGT-TGCCTTCGAGGGCGG3′) and the 3′ primer that introduced a terminal Xho1 site as well as an internal BglII site but retained the correct amino acid sequence (5′CTCGA-GTTACTTAATGTTCTTCAAGATCTCGTC3′). The ΔW construct was produced using the same 5′ primer as described above and the 3′ primer (5′CTCG-AGTTACTGGGCGATGGGCCGGCG-3′) that again inserted a terminal XhoI site but also introduced a UAA termination codon that would terminate translation of the C2B domain immediately prior to the WHTL motif. The GST-ΔH was generated by PCR using the same 5′ primer and 3′primer (5′CTCGAGTCCGG-TGCCCATGCAGCCA3′) again inserted a terminal XhoI site but also introduced a UAA termination codon that would delete helix A, helix B and β-strand 8 of the C2B domain. The GST-ΔK was generated using 5′ primer (5′GAATTCTGCACCCTCAACCCTTACTA3′) and 3′ primer (5′CTCGAGTTACTTCAT-GTTCTTCGCGATCTCGTC) to amplify residue C386 to K474. The GST-D3,4N was created by two step PCR amplification using pGEX/C2B as template DNA. The 5′ end was amplified using primer 5′ primer (5′ACCGTCGTGAACTACAACCGTATTGGCAACCTCC3′) and 3′ primer (5′GAGCTGCATGTGTCAGAGG3′). The 3′ end of GST-D3,4N was amplified using 3′ primer (5′GGAGGTGCCAATACGGTTGTAGTTCACGACGGT3′) and 5′ primer (5′CCAGCAAGTATATAGCATG3′). Overlapping PCR was used to generate the full length D3,4N construct using 5′ primer (5′CCAGCAAGTATATAGCATG3′) and 3′ primer (5′GAGCTGCATGTGTCAGAGG3′). The µ-homology domain (µHD) of STNB was amplified using 3′ primer (5′CTCGAGCAGCACCTAGCCAACTTA3′) and 5′ primer (5′GAATTCCGA-GAGCGAGCGTTGACA3′). The DNA sequences encoding the coiled-coil domain (CCD) of STNA and the µHD of STNB were inserted into the pMAL-c2x vector, and expressed in E. coli as MBP tagged proteins as previously described E. coli to generate MBP-C2B.SYT-1 C2A and C2B constructs were generated in the pGEX-4T-1 vector and expressed as GST-tagged proteins in E. coli lysate and known to alter the properties of recombinant C2B, GST-C2B and MBP-C2B fusion proteins bound to resin were washed twice in 10x volume of 20 mM MES pH 6.3, 20 mM CaCl2, and 0.6 M NaCl (1h on rotating wheel at 4°C), 10x volume of cold TBS, 10x volume of Tris-CaCl2 buffer (1h on rotating wheel at 4°C) and followed by 10x volume of cold TBS, adapted from Ubach et al MBP and GST fusion proteins were produced from cultures according to the manufacturers' instructions (NEB and Amersham), and bound to amylose resin (MBP fusions) or glutathione resin (GST fusions). To remove contaminating nucleic acids present in the http://www.bmm.icnet.uk/~3djigsaw/) using the NMR structure of the C2B domain of rat Synaptotagmin-1 (PDB ID 1tjx) as template The three dimensional model structures of C2B and its variants  were generated using the 3D-JIGSAW comparative modeling server  or 1mM EGTA (EGTA binding buffer). The beads were then collected by centrifugation, and washed twice with 1ml of binding buffer, followed by 1 ml of cold TBS. The complex of MBP-STNA/STNB and GST-C2B was eluted with 20mM glutathione and analyzed by Western Blot using anti-MBP antibody (New England Biolabs) using Amersham ECL™ Western Blotting System. The amount of MBP-STNA/STNB proteins was then quantified by densitometry using ImageJ or Fuji MacBas software programs and the measured intensity was normalized according to the eluted concentration of GST-SYT proteins visualized by Ponceau S stain. The quantified band density was then normalized to value of binding of respective protein in the presence of EGTA, unless indicated otherwise. For investigating the effect of free Ca2+, the GST-C2B was buffered with 1mM EGTA and Ca2+ added to yield the indicated free Ca2+ concentrations using a pKd value of 10.995 for EGTAPull down assays were conducted for 2 h at 4°C using 1.5 µM purified GST proteins as bait on 50 µl glutathione resin and 0.25 µM MBP-STNA and MBP-STNB proteins as the prey protein. The pull downs were carried out on a rotating wheel, for 2 h at 4°C, in a total volume of 500 µl of 10 mM HEPES buffer pH 7.4, 1 mM MgCletal.2 and then 45 min at 4°C. Composition of liposomes were prepared as described previously, consisted of 60%PC (phosphatidylcholine), 20%PE (phosphatidylethanolamine) and either 20%PS (phosphatidylserine) or a further 20%PC Liposomes were prepared as described by Zhang DYDRIGTSE) which contains the aspartate residues  at loop 3 of the SYT-1-C2B domain was added at the same time as addition of MBP-tagged Stoned proteins to the GST-C2B domain as described above in the protein-protein interactions assay. Besides pC2B1, pC2B1.M (Biotin-KICLVVTVVDADRIGTSE) and p-CTRL (Biotin-SINPHLGPLQISLGR) were also tested. The binding assays were performed as described above in Ca2+ binding buffer.The peptide pC2B1 . Independent experiments were done in the presence of 1 mM Ca2+. The bar graph represents the amount of quantified MBP-Stoned proteins normalized against the amount of eluted GST-C2A or GST-C2B proteins. Each value represents mean ± S.E.M.(TIF)Click here for additional data file.Figure S2Molecular Dynamics (MD) simulation of C-terminal truncations and D3,4N of C2B. The RMSD of the structures during 100 ps MD simulation at 300 K suggested all of the C-terminal truncations and D3,4N of C2B examined have similar structural integrity compared to the full-length C2B. The MD simulation data suggested that the deletion/mutation did not affect the protein structure and indicated that all constructs tested are as stable as the wild type. Therefore, the changes in biochemical properties reflect the importance of each region deleted or changed by the mutation.(TIF)Click here for additional data file.Figure S3The effect of pC2B.M peptide on STNB binding. Increasing concentration of random peptide pCTRL has no effect on MBP-STNB binding to GST-C2B. Similar to pC2B1, addition of pC2B.M reduced the MBP-STNB binding. The experiment was performed in the presence of 1 mM CaCl2. The upper panel of Western Blot image shows the MBP-STNB bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red staining of GST-C2B protein of the respective blots.(TIF)Click here for additional data file.


Escherichia coli adenylate kinase (ADK) is a monomeric phosphotransferase enzyme that catalyzes reversible transfer of phosphoryl group from ATP to AMP with a large-scale domain motion. The detailed mechanism for this conformational transition remains unknown. In the current study, we performed long time-scale molecular dynamics simulations on both open and closed states of ADK. Based on the structural analyses of the simulation trajectories, we detected over 20 times conformational transitions between the open and closed states of ADK and identified two novel conformations as intermediate states in the catalytic processes. With these findings, we proposed a possible mechanism for the large-scale domain motion of Escherichia coli ADK and its catalytic process: (1) the substrate free ADK adopted an open conformation; (2) ATP bound with LID domain closure; (3) AMP bound with NMP domain closure; (4) phosphoryl transfer occurred with ATP, and AMP converted into two ADPs, and no conformational transition was detected in the enzyme; (5) LID domain opened with one ADP released; (6) another ADP released with NMP domain open. As both open and closed states sampled a wide range of conformation transitions, our simulation strongly supported the conformational selection mechanism for Escherichia coli ADK. Escherichia coli adenylate kinase (ADK) is a monomeric phosphotransferase enzyme regulating energy homeostasis in cells by catalyzing reversible transfer of a phosphoryl group from ATP to AMP. The structure of ADK is well studied in the past few years, and by now more than 20 crystal structures of ADK from Escherichia coli and other organisms in the absence and presence of substrates have been released in the protein structure databases. According to these crystal studies, the structure of ADK is mainly composed of three major components  was solvated in a simulation box with explicit TIP3P water molecules. To neutralize the system, 4 sodium ions were added to random place 4 water molecules in the simulation box. The atoms of the enzyme were parameterized by Amber force field parameters [5A was done by Antechamber module [ Owing to playing an important role in cellular energy homeostasis, many good attempts have been made to study the three-dimensional (3D) structure of ADK. By now, 29 crystal structures of ADK and its homolog, in the absence and presence of substrates, are available in the protein structure databases –45. In ty Delphi , 48 as ay Delphi  based onrameters , while Ar module  in Amber−6 was applied to constrain all bonds in both simulation systems [−5/bar for solvent simulations [Two simulation systems were involved in this study, with the crystal structures (PDB ID: 4AKE and 1AKE) as initial structures for the open and closed conformations, respectively. Both systems were subjected to the steepest descent energy minimization (~5000 steps) followed by conjugate gradient energy minimization for the next 5000 steps and subsequently equilibrated with the enzyme atoms (or ligand in the closed system) fixed for a short-time molecular dynamics simulations at 300 K to reduce the van der Waals conflicts. Finally, 10-nanosecond (ns) molecular dynamics simulations were performed for both systems under a constant temperature (300 K) by Amber 11 package  with per systems , 53, and systems , 55. Theulations , 57. Theulations , 59. Theα atoms from both open (4ake.pdb) and closed (1ake.pdb) conformations of ADK along all the molecular dynamics simulation trajectories. As shown in  As ADK has been well studied, a lot of kinetic and thermodynamic experimental data were released –64, baseEscherichia coli ADK, we also measured the domain motion of ADK using the geometric center distances of NMP and LID domains with CORE domain along the molecular dynamics simulation trajectories. In the crystal structures, the geometric center distances of NMP and CORE domains in the open (4ake.pdb) and closed (1ake.pdb) conformations were 62.7 Å and 18.4 Å, while those for LID and CORE domain were 70.1 Å (open state) and 21.0 Å (closed state), respectively. As shown in To confirm the conformational transitions for 2+–ATP + AMP → Mg2+–ADP + ADP by Escherichia coli ADK [Escherichia coli ADK. According to the previous theoretical studies, LID domain motion was believed to precede NMP domain motion [Escherichia coli ADK (α2 helix toward α4 helix of CORE domain by nearly 15 degrees with respect to α3 helix, and the enzyme would adopt a complete open conformation (model a in  The large-scale domain motions were detected in the catalytic conversion of Mgcoli ADK , 66. Thun motion , 33, 66.n motion . Anothercoli ADK . The subcoli ADK . The clocoli ADK . With ATcoli ADK . When ancoli ADK . SubsequOur proposed mechanism was also supported by some experimental results. Firstly, the complete closed conformation of ADK was found to be the only product-forming state . In otheFurthermore, our proposed mechanism was also in good agreement with previous theoretical studies. In 2008, Lu and Wang developed a coarse grained two-well model to study the conformational dynamics of ADK in microscopic detail . They idEscherichia coli ADK, which can sample a large set of conformations between the open and closed states. Our computational results strongly supported the conformational selection model proposed for ADK [For the conformational transitions in proteins or enzymes, two different substrate binding mechanisms, induced-fit model, and conformational selection model can be proposed. The former is a model for enzyme-substrate interactions to describe that only the proper substrate is capable of inducing the conformational changes in the binding site, allowing the enzyme to perform its catalytic function . The lat for ADK , 70–74.Escherichia coli ADK, we performed long time-scale molecular dynamics on both open and closed states of ADK. The two-dimensional RMS deviations of the Cα atoms from both open and closed conformations along simulation trajectories confirmed the fact that conformational transitions between the open and closed conformation occurred during our simulations. Additionally, two significant intermediate states were identified by monitoring the domain distances between LID/NMP and CORE domains, one of which adopted an open LID domain with a closed NMP domain, and the other one employed an open NMP domain with a closed LID domain. Based on these computational results, we proposed a possible mechanism for the large scale conformational transitions and the catalytic function (Escherichia coli ADK.In conclusion, to study the large-scale domain motions in function . The pro


Drug-induced lupus erythematosus (DILE) is a lupus-like syndrome temporally related to continuous drug exposure which resolves upon drug discontinuation. There are currently no standard diagnostic criteria for DILE. Findings include skin manifestations, arthritis, serositis, anti-nuclear and anti-histone antibodies positivity. Similarly to idiopathic lupus erythematosus, DILE can be divided into systemic (SLE), subacute cutaneous (SCLE) and chronic cutaneous lupus (CCLE). Systemic DILE presents as a milder version of idiopathic SLE, and the drugs most frequently implicated are hydralazine, procainamide and quinidine. Anti-TNFα therapies are the latest class of medications found to be associated, although rarely, with a “lupus-like” syndrome, which is however clinically distinct from classical DILE. Drug-induced SCLE is the most common form of DILE. It is very similar to idiopathic SCLE in terms of clinical and serologic characteristics. The most commonly implicated drugs are antihypertensive drugs and terbinafine, but in recent years also proton pump inhibitors and chemotherapeutic agents have been associated. Drug-induced CCLE is very rare and usually caused by fluorouracil agents and NSAIDS, but some cases have induced by pantoprazole and anti-TNFα agents. Systemic lupus erythematosus (SLE) is a common autoimmune disease, with an incidence in Europe and North America varying between 1 and 10 cases per 100 000 per year . It has There are currently no standard diagnostic criteria for DILE, and in many cases patients with DILE do not fulfill the American College of Rheumatology (ACR) criteria for SLE. The four most common features  could be employed as diagnostic criteria; in addition the symptoms must have begun after initiation of the treatment with a drug and must resolve after discontinuation .The pathogenesis of DILE remains unclear, and available data strongly suggest that there is no single mechanism responsible for the induction of autoimmunity by all lupus-inducing drugs. DILE does not present with the features of a typical drug hypersensitivity reaction. In particular, there is no evidence of drug-specific T cells or antibodies; the reaction occurs frequently months or years after exposure; development of DILE depends on the cumulative dose, and the recurrence of symptoms after rechallenge generally takes 1–2 days, indicating the absence of immune sensitization to the culprit drugs. Lupus-inducing drugs are commonly metabolized (oxidized) to reactive species by activated leucocytes, thus acquiring the capacity to bind to carrier proteins and become immunogenic. Alternatively, reactive drug metabolites could directly cause cell death via a non-immune mediated process or could alter degradation and clearance of apoptotic cells which leads to the loss of tolerance to self antigens. Disruption of central immune tolerance has also been hypothesized . FinallySimilarly to idiopathic lupus, DILE can be divided into systemic (SLE), subacute cutaneous (SCLE) and chronic cutaneous lupus (CCLE), both in the form of discoid and tumidus (LET).Systemic DILE usually resembles a milder version of idiopathic SLE . It is rOther nonspecific skin features, including urticarial vasculitis, livedo reticularis and skin ulcers, may be part of the clinical presentation of systemic DILE . TypicalDrug-induced SCLE is the most common form of DILE, with at least 128 cases reported in the English language literature . It presDrug-induced CCLE is rare and usually refers to fluorouracil agents and NSAID . MoreoveAnti-TNFα therapies are the latest class of medications found to be associated with a “lupus-like” syndrome . Most ofIn contrast to other forms of DILE, induction of ANA and/or anti-DNA antibodies in patients treated with anti-TNFα therapy is well established and quite common  Table 3Table 3. The mechanisms by which anti-TNFα therapy induces lupus remain unclear but are likely to differ from classic DILE. One hypothesis is that TNF inhibitors interfere with normal cell apoptosis, leading to a decreased clearance of autoreactive T and B cells and cellular debris, including nuclear material. Accumulation of nucleosomes and their breakdown products in a genetically susceptible host may result in the development of autoantibodies. Another hypothesis is that the suppression of the T-helper type 1 response by TNF blockers could enhance a T-helper type 2 response leading to SLE. Finally, bacterial infections which are increased with TNF blockers, may induce polyclonal B-lymphocyte activation and favor autoantibody production .DILE is a reversible lupus-like condition due to exposure to an increasing number of drugs. Its symptoms are generally mild to moderate with resolution of both clinical and serological features over time following drug discontinuation. The possibility of drug-induction should always be considered in all patients with lupus erythematosus, because of the easy reversibility of the lesions. The management of DILE consists mainly of the discontinuation of the implicated drug. For severe or refractory cases the addition of systemic corticosteroids at the doses commonly used for the idiopathic form is the first-choice therapy. Some patients may need additional immunosuppressive therapy, including azathioprine, cyclophosphamide, methotrexate or mycophenolate. In the case of anti-TNFα DILE, if the clinical presentation of drug-induced lupus is mild and well tolerated, TNFα inhibitors do not need to be discontinued. The appearance of ANA is not a reason for stopping TNFα inhibitors in asymptomatic patients with psoriasis. There is limited evidence to support the switch to alternative TNFα antagonists in patients who develop anti-TNFα DILE .None.


T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed.The crystal structure of the N-terminal part of  Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from  The subsequent substitution of four residues with methionine (construct DnaJΔ108–114/4M) allowed SeMet-SAD phasing, resulting in the determination of the first crystal structure of a complete functional DnaJ containing J, GF and C-terminal domains to 2.9 Å resolution. This structure was then used as a search model for molecular replacement with data from the wild-type DnaJ crystals, which indicated a large degree of flexibility between the GF and C-terminal domains, which was then investigated by EPR spin-labelling, simple modelling and cross-linking experiments, resulting in a hybrid model of the highly dynamic DnaJ molecule.Here, we describe an in-depth structural characterization of 2.2.1.NcoI and NotI (New England Biolabs), purified by agarose-gel electrophoresis and ligated into pET24d. For the DnaJΔ108–114/4M construct, the region coding for residues 108–114 was deleted and residues Leu57, Ile149, Leu180 and Leu233 were mutated to methionine because of their aliphatic nature.Constructs were prepared by either QuikChange or two-stage PCR. After two-stage PCR, the PCR products were ligated into pCR2.1TOPO (Invitrogen) and positive clones were selected using blue/white screening. Correct clones as identified by sequencing were digested with E. coli BL21(DE3) cells and were purified essentially as described in Klostermeier et al. (1999114) or by a combination of heat denaturation and Ni2+-affinity and gel-filtration chromatography (other constructs). DnaJTth114 was expressed in E. coli BL21(DE3) cells and precipitated from a cell-free extract by adding solid ammonium sulfate to 80% saturation at room temperature, followed by stirring for 30 min at 277 K. The precipitate was recovered by ultracentrifugation (30 min at 277 K at 65 000g) and dissolved in buffer B . Further contaminants were heat-denatured in a water bath at 348 K for 30 min, followed by removal of the precipitated material using ultracentrifugation . The supernatant was loaded onto a Q Sepharose column equilibrated with buffer B. DnaJTth114 was found in the flowthrough and its purity was assessed by SDS–PAGE. When required, the protein was further purified on a Superdex 75 column in buffer C . The mass of the protein as determined by MALDI–TOF MS was 12 902.7 Da, which is nearly identical to the calculated average molecular mass of the construct minus the N-terminal methionine residue (12 903.4 Da).All DnaJ constructs were heterologously expressed in  al. 1999 either bE. coli BL21(DE3) cells were transformed with the appropriate plasmid and grown on 3–6 l LB medium. For the production of SeMet-DnaJΔ108–114/4M, 5 l minimal medium supplemented with 50 mg l−1l-selenomethionine at 310 K was used  and a Complete EDTA-free protease-inhibitor cocktail tablet was added. The cells were lysed by two passes through a fluidizer (690 kPa), and the lysate was incubated in a water bath at 343 K for 10 min and then cooled on ice. The mixture was centrifuged for 40 min at 30 000 rev min−1 in a Beckman Ti60 ultracentrifuge rotor (277 K) and the supernatant was mixed with 12 ml 50% Ni–NTA agarose in buffer D. The resulting suspension was gently agitated for 2 h at 277 K and then poured into a 6 ml column. This column was washed with 60 ml buffer E . The protein was then eluted with 12 ml buffer F . The protein was concentrated by ultrafiltration to 3 ml and submitted to a 340 ml Superdex 200 column equilibrated with buffer G  running at 2 ml min−1. Pure fractions as judged by SDS–PAGE analysis were pooled and then washed and concentrated by ultrafiltration in buffer H  to an A2801 cm of ∼9–14. The protein was flash-cooled in liquid nitrogen and stored at 193 K.For the production of most of the other constructs, 2.2.2.2.1.114 was crystallized by equilibrating hanging drops consisting of 2 µl 17 mg ml−1 DnaJTth114 in buffer B plus 10 mM SrCl2 and 2 µl 35% PEG 1500 against a 600 µl reservoir of 35% PEG 1500 in Linbro plates. Crystals were cryoprotected in 35% PEG 1500, 10% PEG 400, 25 mM Tris–HCl pH 7.5, 10 mM SrCl2. All data were processed with XDS . Inclusion of the 2.9 Å resolution data and solvent flattening with DM resulted in an improved map into which the structure was built by iterative cycles of rebuilding using Coot  and 4j80 (DnaJΔ108–114/4M)A 3.2 Å resolution SAD data set was collected from a crystal grown in the presence of 2 m2.2.3.H with an A2801 cm of ∼9–14 plus 2 µl reservoir solution and 0.2 µl 24 mM CYMAL-5  against 600 µl reservoir solution consisting of 1.0 M (NH4)2HPO4, 0.1 M Tris–HCl pH 8.5. The resulting box-shaped crystals were dehydrated overnight in a saturated solution of trimethylamine oxide (Sigma) in reservoir solution, which served as both a dehydrating agent and a cryoprotectant. Typically, the diffraction resolution improved to around 4 Å after 4 h of dehydration. Dehydration for longer than 20 h reduced the resolution. The best data (3.8 Å resolution) were obtained at the SLS from a crystal which had been soaked for 16 h in 3 mMp-chloromercuribenzoate in the dehydration solution. The resulting data were phased by molecular replacement with Phaser  DnaJ crystals were produced using the sitting-drop method by equilibrating 2 µl protein solution in buffer 2.3.et al., 1987S-methyl methanesulfonothioate; Pannier et al., 2000For spin labelling of DnaJ variants  were dialysed for 16 h against 1 l buffer I . For cross-linking, fresh proteins were diluted to 10 µM in 30 ml buffer J . To these solutions, an equimolar amount of BMB , i.e. 1 mol DnaJ dimer:1 mol BMB, was added as follows: 30 µl of 10 mM BMB in DMSO was added in ten steps of 3 µl with vigorous stirring at 5 min intervals. After the last addition the solutions were left to incubate for a further 1.5 h while stirring.The proteins . From each mutant, a fraction at the end of the dimer peak (i.e. free from tetramer contamination) was investigated using reducing SDS–PAGE to check for the presence of covalently cross-linked DnaJ dimers.To separate DnaJ dimers from DnaJ tetramers generated by interdimer cross-linking, the proteins were concentrated to 100 µl and submitted to a 23 ml Superdex 12 (10/300) column  running at 0.8 ml min2.5.MatLab using simple models of spherical amino acids. Starting from the crystal structure, an ensemble of conformations was calculated using a Monte Carlo algorithm employing a pseudo-energy term calculated from the EPR distance distributions and the number of clashes between residues. Details are included in Appendix CComputational modelling of the mobility of DnaJ molecules was performed in 2.6.et al. , comprising the J and GF domains, and determined its structure to 1.64 Å resolution , which are highly similar . After helix IV, the polypeptide chain folds back onto the protein at the Pro6 motif, which adopts a polyproline II conformation , which was shown to be functional in refolding assays . Crystallization of this variant allowed structure solution to 2.9 Å resolution using SeMet phasing with a construct containing four additional methionines (DnaJΔ108–114/4M), resulting in a structure containing all three domains .In the DnaJns Fig. 3a.3.3.Δ108–114 dimerizes via its C-terminal domains, resulting in a V-shaped molecule with the J/GF domains at the ends of the two stalks  like feet on a pair of legs. The J and GF domains interact tightly through a hydrophobic interface involving six of the seven phenylalanines of the GF domain and helix III of the J domain . The same interaction is seen in all six independent copies of the J/GF domains in the DnaJ114 structure. In all four monomers in the asymmetric unit of the DnaJΔ108–114 crystals the orientation of the C-terminal and J/GF domains differs, showing that the Δ108–114 deletion does not restrict their mutual motion. Moreover, the J/GF regions of chains B and D showed poor electron density, also indicating flexibility.DnaJks Fig. 1b like fin Fig. 1c. The sΔ108–114 only the C-terminal domain was observed , indicating a high degree of mobility of the J and GF domains. Indeed, the packing of the wild-type DnaJ crystals leaves sufficient space for the J and GF domains to attain a different orientation with respect to the C-terminal domain in each of the four monomers in the asymmetric unit. Analysis with DynDom  between single cysteine mutations placed in the J/GF domains of DnaJΔ108–114. This resulted in covalently cross-linked DnaJΔ108–114 dimers, showing that in solution the J/GF domains within a DnaJΔ108–114 dimer can approach each other to within 11 Å, which, given the structure of DnaJΔ108–114, would seem to be possible only when significant disorder exists in solution.Given previous findings of predominantly disordered GF domains . Strikingly, these revealed intramonomer distances (in the 20–30 Å range) which excellently match the distances derived from the crystal structure . The large modulation depth in the first 250 ns of the time traces governed by these short distances shows that in solution the vast majority of DnaJ molecules adopt the same J/GF structure as observed in the crystal structure. Appendix BTo check whether this disorder in solution occurs in the GF domain, four-pulse ELDOR (pELDOR) distance measurements  which were not expected from the crystal structure, where intermonomeric distances are only observed at 80 Å and above owing to the pronounced V shape of the dimer. Still, these distances indeed stem from intermonomer dipolar interaction as they are also observed in control experiments on two singly labelled DnaJ variants carrying only one spin label per monomer . The broad distance distribution over the whole experimental range shows tails (with low probability down to 20 Å), which is consistent with the cross-linking results and confirms high structural variability in parts of the DnaJ protein backbone in solution.However, additional pELDOR signal contributions occur in the 40–60 Å range  the sum of all Cα–Cα distances smaller than 5 Å and (ii) a term calculated from the EPR distance distribution using the Boltzmann distribution formula. Using these pseudo-energies, Monte Carlo simulations were performed to obtain ensembles of plausible conformations for all models, which were then checked for their consistency with the cross-linking results by checking for the occurrence of conformations in which the cross-linking sites were within 12 Å of each other during a Monte Carlo run of 20 000 trial structures.To visualize how much flexibility would be needed to account for the observations described above, we prepared simple geometric models of spin-labelled DnaJCΔ108–114, despite the obvious reduction in interdomain flexibility caused by the deletion of seven residues in the J/GF linker, rotation around the backbone bonds of residues 103–110 alone, at the hinge between the J/GF and C-terminal domains, already suffices to explain the cross-linking results.The calculations (see Appendix 3.7.b).The various mutations produced for cross-linking experiments were also evaluated for their ability to assist in luciferase refolding by DnaK. Importantly, not only are all of the mutants employed still active, they also display significant modulation of the luciferase refolding activity, in particular when the mutations are located in the GF domain Fig. 4b.4.T. thermophilus DnaJ the GF domain adopts a highly ordered structure both in the crystal and in solution. Nonetheless, T. thermophilus DnaJ is a flexible molecule as shown by cross-linking and EPR studies as well as by the lack of density for the J and GF domains in the DnaJwt crystal structure. Since the J/GF domains visible in the DnaJΔ108–114 crystal structure each assume a different orientation relative to their respective C-terminal domains, it seems likely that this flexibility is, at least to a large extent, concentrated at the junction between the J/GF domain and the C-terminal domain. Indeed, our very simple molecular modelling studies showed that flexibility in this region alone is sufficient to explain the EPR and cross-linking data. Possibly, the effects that mutations in the GF region have on luciferase refolding are caused by a modulation of this flexibility. In support of this hypothesis, one may compare the T. thermophilus and E. coli DnaK–DnaJ systems. In E. coli DnaJ, which stimulates DnaK ATPase activity strongly, a highly flexible GF domain was observed in NMR studies, although a partial structure was observed  were surmised (Huang, Flanagan et al., 1999T. thermophilus DnaJ, which stimulates DnaK ATPase activity only weakly, the structures presented here indicate a strongly ordered GF domain.Given the structural information available previously, the GF domains of DnaJs have frequently been seen as highly disordered flexible structures. The structures and EPR distance distributions of DnaJ presented here necessitate a departure from this view, as they show that in et al., 2007et al., 2011et al., 2012A possible explanation could lie in the structure of the DnaK–DnaJ complex. If, as suggested (Han & Christen, 2003DnaJ, 4j7zPDB reference: 4j80PDB reference:


Identifying domains in protein sequences is an important step in protein structural and functional annotation. Existing domain recognition methods typically evaluate each domain prediction independently of the rest. However, the majority of proteins are multidomain, and pairwise domain co-occurrences are highly specific and non-transitive.Plasmodium falciparum, for which over 38% of the genome is currently unannotated. Our approach enables high-confidence annotations in this organism and the identification of orthologs to many core machinery proteins conserved in all eukaryotes, including those involved in ribosomal assembly and other RNA processing events, which surprisingly had not been previously known.Here, we demonstrate how to exploit domain co-occurrence to boost weak domain predictions that appear in previously observed combinations, while penalizing higher confidence domains if such combinations have never been observed. Our framework, Domain Prediction Using Context (dPUC), incorporates pairwise "context" scores between domains, along with traditional domain scores and thresholds, and improves domain prediction across a variety of organisms from bacteria to protozoa and metazoa. Among the genomes we tested, dPUC is most successful at improving predictions for the poorly-annotated malaria parasite http://compbio.cs.princeton.edu/dpuc/. Pre-computed results for our test organisms and a web server are also available at that location.Overall, our results demonstrate that this new context-based approach will provide significant improvements in domain and function prediction, especially for poorly understood genomes for which the need for additional annotations is greatest. Source code for the algorithm is available under a GPL open source license at  Protein domains are fundamental units of protein structure, function, and evolution. As a result, domain prediction is an important first step in the annotation of protein sequences . EnhanceThe majority of proteins contain more than one domain . DomainsHere, we exploit the tendency of certain domains to co-occur with each other in order to improve domain identification. While domains within any given sequence are typically identified by considering each domain family individually, domain co-occurrence or "context" is useful in detecting weak sequence similarity . In partDomain Prediction Using Context), uses Pfam . Finally, observed pairs with negative scores are instead set to zero. This way, observed pairs are never penalized. For Pfam 23, only 5 out of the 15,929 observed pairs had negative scores that were subsequently set to zero. We also experimented with other log-odds scoring schemes  runs in 96.4 ± 41.4 seconds per protein (mean ± standard deviation), while the dPUC overhead  runs at an additional 0.0229 ± 0.865 s/protein. However, the runtime distributions have very long tails, as can be seen in Additional File Since each protein sequence is a separate problem, the problem can be parallelized over proteins. We considered the 25,047 proteins of E-value" method uses domain E-value thresholds instead of the GA thresholds.We tested three non-context methods that serve as natural baselines for dPUC. The first is the "Standard Pfam", which uses their curated and domain-specific "gathering" (GA) thresholds  on real and shuffled protein sequences. The key idea is that domain predictions on shuffled sequences arise by chance alone, whereas predictions on real sequences give us the total number of predictions , and their ratio approximates the false discovery rate. We shuffle the residues of each protein separately, thereby preserving the amino acid composition of each sequence as well as the length distribution over each proteome. For the context methods , we run them on the real sequence concatenated to its shuffled sequence, but only count the number of predicted domains on the shuffled portion of the sequence. Therefore, we count the random predictions that might have been rewarded by positive context not only with other random predictions, but also with potentially real domains from the real sequence Figure . The shuA be the number of predictions per shuffled protein, and let R be the number of predictions per real protein. Then FDR = A/R, which is a common approximation of the FDR [An and Rn be the quantities defined above when we use negative context only, and A and R when we use the E-value threshold of interest for candidate domains. The difference of the data corresponds almost entirely to the novel domains, ignoring the effect of the initial negative context elimination:We estimate the false discovery rate (FDR) as follows. Let  the FDR . To calcet al. [This benchmark is only appropriate for methods in which the orientation of the domains is unimportant, as it is for the methods tested here. We note that the Markov model of Coin et al.  is sensiPlasmodium species' proteins because their sequence divergence is large enough to discard "coherent" false predictions due to high sequence similarity, yet the sequences are similar enough for orthologs to be identified easily, and domain architectures are largely conserved. Moreover, as we show below, our approach performs well in improving domain identifications in these species.In our second test, we measure how often domains are predicted across orthologs. The key assumption is that real domains are very likely to be present in orthologs, whereas false domains are very unlikely to be. Therefore, the average "ortholog coherence" score is inversely related to the amount of spurious predictions. We chose the Plasmodium species using OrthoMCL 1.4 [Plasmodium species . This left us with 5523 groups with a total of 30,065 proteins. Each group was aligned with T-Coffee 8.14 [We computed the orthologous groups of six oMCL 1.4 . We obtafee 8.14 , using tfee 8.14 , and Musfee 8.14 . The scoD. melanogaster, C. elegans, S. cerevisiae, E. coli, and several pathogens including the eukaryotic human malaria parasites P. falciparum and P. vivax, and the prokaryotic parasite M. tuberculosis. We test our method separately on each of these organisms since they have different and sometimes extreme biases in protein lengths, amino acid compositions, amino acid coverage by domains, and domain family content  organism than they are for model organisms. Note that even when limiting the dPUC candidate domains to those predicted by the Standard Pfam , we see a sharp decrease in dPUC's FDR relative to the Standard Pfam; this improved performance can be directly attributed to the removal of false predictions using negative context scores.For a given method , we estimate its FDR as the ratio of the number of predictions made on shuffled sequences , to the number of predictions made on the real sequences only. This concatenation approach allows noise in the shuffled sequence to be boosted by potentially real domains via context Figure . We testd Figure . The StaE-value thresholds on the candidate domains, the FDR is the net effect of removing domains from the Standard Pfam through negative context, as well as adding new domains with positive context. Since negative context alone reduces the FDR of the predictions, the FDR of the new domains must be larger than the FDR of the whole. For dPUC with E ≤ 1, the FDR of the new domains only (see Methods) varies between 0.8-2%, depending on the organism  in which a pair score is logarithmically proportional to how often the domain family pair is observed in Standard Pfam. Although these log-odds scores seem naturally compatible with the HMMER log-odds scores, it is not certain that this is the form the scores should have. To test the importance of these scores, we shuffled the positive scores (three times), which resulted in decreased performance compared to our original context scores, but better performance than that of non-context methods (data not shown). Therefore, carefully choosing values for our scores is important, and our form, which weighs the evidence of co-occurrence per domain family pair, performs better than random scores from the same distribution. Additional score parameter variations are discussed in Additional File E ≤ 1 to identify the candidate domains, which corresponds to an overall FDR of 0.03-0.2% across organisms; that is, this is within an order of magnitude of the Standard Pfam FDR of 0.01-0.09%  or domain removal due to negative context. In either case, we expect the new domain predictions to be more accurate, and the resulting removal of GO terms is welcomed. Interestingly, the addition of domains can also lead to GO term loss, which is a consequence of the MultiPfam2GO probabilistic model combined with incomplete training data  with orthologs or in-paralogs in these organisms as predicted by OrthoMCL, and plotted these scores against their number of domain predictions per protein for the same methods tested earlier . Three predictions find direct support in the literature. PF11_0086, the predicted "poly(A)-binding protein-interacting protein 1" PAIP1, has a strong yeast 2-hybrid interaction with PFL1170w [P. falciparum. Additionally, the two DEAD-box helicases PFE1390w and MAL8P1.19, predicted to be the orthologs of ABSTRAKT and DBP10 respectively, agree with the assignments of a recently published curated list of P. falciparum helicases [Careful manual analysis of novel domain predictions on the re Table  or due tre Table . Our disPFL1170w , the annelicases . Such neelicases  as commuWe have shown that domain identification can be greatly improved across a diverse set of organisms by exploiting domain co-occurrence information. Our method is more successful at increasing domains in genomes with lower domain coverage , at the cost of an exponential (in k) increase in parameters, which become difficult to estimate accurately and increase the computational requirements of the approach. In contrast, dPUC allows interactions between all domains, as opposed to just the previous k domains, while keeping the parameter space small, simplifying parameter estimation and reducing our program's memory usage. Furthermore, the source code accompanying our work should facilitate further improvements in context-based identification of domains.To our knowledge, there has been limited prior work examining the utility and systematic use of domain context for domain prediction , and to P. falciparum if high scoring domains that preferentially appear with them are found in the same sequence [E-value thresholds since this is much more prone to false predictions. Fourth, our problem is combinatorial in nature, allowing domains with low E-values to overcome higher E-value candidates if the context is more favorable, while CODD simply prioritizes candidate domains by E-value. Fifth, CODD uses a limited positive context network that does not include repeating domains and eliminates pairs that do not have small enough p-values derived from the hypergeometric distribution. Although our benchmarks show that negative context and our more complete dPUC positive context networks are critical differences  that uses a list of "favored" domain pairs to predict lower-scoring domains in sequence . We haveOther works, such as AIDAN  and a siDomain context is independent of the information that HMM scores capture, since an HMM score only uses the sequence the domain encompasses, ignoring the surrounding sequence that contains the rest of the domains, while domain context allows these other domains to affect each other. These two information sources do not overlap. In other words, HMMs capture so-called "vertical" information  while doWe have implemented a log-odds system to score the co-occurrence of domain pairs. Indeed, log-odds scores are widespread in bioinformatics, and our scoring system is inspired by the log-odds scores used in local sequence alignment (LSA) . We borrWhile we applied dPUC to Pfam using HMMER2, there are other tools available which could be used, including SAM  and the Plasmodium falciparum, the parasitic agent of malaria, for which much of the core cellular machinery remains unidentified. Overall, our approach is likely to be most useful for poorly understood genomes where the need for additional annotations is arguably the greatest.Common domain identification methods typically consider each domain separately, and they have limited applicability when the similarity between the query sequence and known domains is very low, as is the case for poorly understood genomes. Here we significantly improved domain predictions by exploiting the tendency for domains to co-occur in specific combinations. We developed an approach based on the observation that weak domain predictions are better supported if they appear in previously-observed combinations, while domain combinations that have never been observed are less likely to be valid. Our method improved domain predictions in all organisms tested, including the best known model organisms. The biggest improvements were seen for the divergent organism AO and MS conceived and designed the approach and experiments. AO implemented the approach and performed the experiments. All authors analyzed the data, wrote, read and approved the final manuscript.Supplementary information. PDF file (17 pages) that includes all the supplementary methods, results, figures and tables.Click here for file


Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5′-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5′-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in  Gluconoacetobacter xylinusCyclic di-GMP (bis-(3′-5′) cyclic dimeric guanosine monophosphate, c-di-GMP) was first considered as an allosteric regulator of cellulose production in in vitro that EAL domains can pursue the hydrolysis of 5′-pGpG into two GMP molecules in vivoThree protein domains, GGDEF, EAL, and HD-GYP, have been reported to be involved in c-di-GMP metabolism. They are named after the conserved amino-acids motifs in these domains 2+ or Mn2+. Conversely, the PDE-A activity is inhibited in presence of calcium ions. The comparison of active EAL domains and of degenerated non-catalytic EAL domains has allowed to identify 14 polar residues important for the PDE activity. Based on the structure of the EAL domain containing protein YkuI from Bacillus subtilis and site directed mutagenesis on the EAL domain containing protein RocR from P. aeruginosa, a mechanism explaining the phosphodiesterase activity by the EAL domains has been proposed 2+ base-assisted catalysis, but the number of Mg2+  necessary for catalysis remains elusive 2+ ions.Bacteria using c-di-GMP signaling networks produce several proteins containing an EAL domain. EAL domains have been reported to be catalytically active in presence of either MgP. aeruginosa is a 76,000 Da protein identified in mutants lacking twitching motility in response to environmental factors Xanthomonas axonopodis, a citrus pathogen, has been reported to interact with PilZ, a protein involved in type IV pilus biogenesis P. aeruginosa contains a CheY-like response regulator domain (REC), a PAS-PAC domain commonly involved in environmental sensing, and one GGDEF and one EAL domain. FimX is located at one pole of the bacterial cell probably by its N-terminal REC domain. The full-length protein possesses a PDE-A activity stimulated by Mg2+ and Mn2+ but strongly inhibited by Ca2+, consistently with what has been described for other proteins with an EAL domain Caulobacter crescentusFimX (PA4959) from Here we solved the crystallographic structure of FimX-EAL bound to 5′-pGpG and show that this domain doesn’t display major structural rearrangements whether bound to 5′-pGpG, c-di-GMP or in apo form. We also probe its affinity for c-di-GMP and 5′-pGpG in solution by NMR spectroscopy and determined which protons of the ligand are recognized by the protein.12121 and P43212 respectively. Crystals diffracted to 2.3 Å and 2 Å resolution respectively (Thiobacillus denitrificans (PDB code 2R6O) as search model. Both structures contain two molecules per asymmetric unit related by a non-crystallographic two-fold axis (The FimX-EAL domain (residues 438–686) was crystallized alone and in presence of the substrate c-di-GMP in space groups P2ectively . The strold axis .3212 crystal form leads us to place a molecule in the active site (3212 space group) will be further referred as the complex form, and that with no density in the active site (P212121 space group) as the apo form.Residual density in the active site of both EAL domains of the asymmetric unit of the P4ive site  A. Densiive site  B. The s6β secondary structure succession ccession . This seFimX-EAL displays very little variations within the asymmetric unit of both crystal forms with r.m.s. deviations of 0.47 Å over 249 superimposed Cα positions and 0.38 Å over 245 superimposed Cα positions in the complex and in the apo forms respectively. The structures of FimX-EAL complex and apo form are also very similar to each other with a r.m.s. deviation of 1.18 Å over 485 superimposed Cα positions.B. subtilis YkuI (chain A) and our structure of P. aeruginosa FimX-EAL bound to 5′-pGpG (chain B) are the most divergent structures, with a r.m.s. deviation rising to 2.59 Å over 220 superimposed Cα positions.Superposition of all known EAL domain structures was performed chain by chain. The core barrel of these two EAL domains is invariable. Deviation contributions arise from helices and loops. Interestingly, loop-8 is the only loop highly superimposable. The closest structure to ours is FimX-EAL solved previously (PDB code 3HV8) 2 with a score of 0.321 for the complex form and is 1430 Å2 with a score of 0.401 for the apo form. In both dimers, the computed scores for the interaction are relatively weak but there is however a large interface area due to hydrogen bonds and hydrophobic contacts. Similar homodimer was already described in the asymmetric unit of crystals in space group P41212 and in the crystal lattice with a symmetry related molecule in space group P6422 FimX-EAL is monomeric in solution whether it is free or bound to c-di-GMP 3 and 3609 Å3 for the complex and the apo forms respectively , the overall position and conformation of the ligand in the active site are different in the two structures  and Glu475 (β2) from the EAL motif (turned EVL in FimX), a water molecule and the 5′-phosphate from 5′-pGpG . The magp-nitrophenyl) phosphate (BpNPP) as substrate in Mn2+ buffer. This compound was proved to be an alternative to c-di-GMP to measure PDE-A activities and was used with several EAL domains like HmsP pNPP isn’t the natural substrate of EAL domains, previous studies proved that a millimolar KM for BpNPP corresponds to a nanomolar KM for EAL domains for the natural substrate c-di-GMP. However the turnover kcat may display strong variation compared to the turnover for the natural substrate c-di-GMP p-nitrophenol was measured for several hours as a function of the BpNPP concentration  and initial rates were determined for each concentration. Full-length FimX displays a KM of 1 mM and a kcat of 6.4.10−4 s−1 (M measured for full-length FimX is lower to the one measured for TBD1265 and SFV_3559 (20 mM and 14 mM respectively). In contrary, kcat is weaker than the one measured for TBD1265 and SFV_3559 EAL domains (0.06 s−1 and 8 s−1 respectively). The catalytic efficiency kcat/KM of full-length-FimX is 10-fold lower than the one of TBD1265. These experiments prove that FimX full-length displays a strong affinity for its substrates but a very weak turnover considering its KM and kcat. FimX has a low activity with BpNPP as substrate. This low phosphodiesterase activity is in agreement with what was already described In order to assess the phosphodiesterase activity of FimX and thus to validate the presence of 5′-pGpG in our crystallographic structure, we performed enzymatic assays using Bis  buffer at 308 K and pH 6.5, for which only small amounts of unlabeled protein and an excess of ligand are required  NMR experiments required . The H2O protein  D and G. protein  B and F. protein  D. This in vitro. Gel filtration and SAXS experiments on FimX-EAL with or without 5′-pGpG could not reveal the formation of a dimer in solution (data not shown). The dimerization interface involves mainly N- and C-terminal extremities residues, and in less extent helices α2, α6 and α7 helices. The N- and C- terminal extremities may be more flexible in solution than in the crystal context. Structural rearrangements of the flexible N- and C-terminus may be necessary to form the oligomer and crystal packing could stabilize these conformations. Because the residues involved in the interface are mostly polar, burying polar residues during dimerization does not have a large contribution to the entropic effect and will not stabilize the dimer.We have crystallized the FimX-EAL in its apo form and in complex with 5′-pGpG as a homodimer. It was previously shown that FimX-EAL bound to c-di-GMP crystallized as a monomer because dimerization would cause a large number of ligand-ligand clashes in the binding pocket. Dimerization of apo FimX-EAL was thought to prevent c-di-GMP binding P. aeruginosa is tetrameric in solution The oligomeric state of EAL domains and full-length proteins containing an EAL domain is very variable and depends on the ligand bound to the domain. Several EAL domains, like YkuI, BlrP1, YcgF and tdEAL have been reported to also form EAL-EAL mediated homodimers in crystals when they are bound to c-di-GMP and for some of them in the apo form. The existence of homodimeric forms was confirmed in solution by analytical ultracentrifugation, size exclusion chromatography and dynamic light scattering FimX has a strong affinity for c-di-GMP according to the Kd (about 120 nM) computed from ITC experiments Interestingly, in the complexes, the 5′-pGpG conformation is very different from that of the c-di-GMP preventing clashes between the two ligands inside the dimeric catalytic pocket. The only position to be conserved is that on the non-hydrolyzed phosphate in 5′-pGpG which occupies the positions of the target phosphate of c-di-GMP in the hydrolysis reaction. 5′-pGpG is thus in position for a potential second round of hydrolysis. Neither the guanines nor the sugars of 5′-pGpG are superimposed on their counterpart in c-di-GMP. Conversely to c-di-GMP, and according to both our crystallographic structure and NMR data, none of the two guanines seem involved in the 5′-pGpG recognition. We performed a sequence alignment of FimX with other EAL domains in order to define which of the ten conserved residues identified as essentials for the catalytic activity B. subtilis YkuI bound to c-di-GMP 2+. After the hydrolysis of the c-di-GMP, the Mg2+ is not located at the conserved position observed in other EAL domains. It remains coordinated by a water molecule, by one of the two oxygens of the hydrolysed phosphate, by a carboxylic oxygen from Glu475 of the EAL motif and by the backbone carbonyl of Gln461. This network of interactions supports a catalytic mechanism in which a water molecule activated by the cation would perform a nucleophilic attack on one of the cyclic phosphates and hydrolyse the c-di-GMP as proposed for YkuI and BlrP1. According to this model, during the reaction, the hydrolysed phosphate would move away from its original position, dragging the Mg2+, and induce conformation changes of the whole hydrolyzed ligand backbone, resulting also in a change of the positions of the bases. The fact that only Glu475, a water molecule and one magnesium ion are essential to carry out the hydrolysis of the c-di-GMP, could explain why the overall structure of the FimX-EAL domain doesn’t undergo major structural rearrangements during the hydrolysis. Interestingly, the position of the hydrolysed phosphate is highly superimposable to the equivalent position Leishmania mexicana TIM in the complex with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (PDB code 1IF2), suggesting the existence of a conserved phosphate-binding motif involving the C-terminal loops of TIM and EAL domains. Our results also suggest that the exact number of cations involved in the catalysis may not be fixed amongst EAL catalytic pockets.The number or coordinated magnesium ions in the catalytic site of the EAL domains varies amongst the structures: in BlrP1 and tdEAL, two magnesium ions are present Xanthomonas proved that FimX homolog is able to bind PilZ, a major protein in type IV pili biogenesis, via its EAL domain We can’t exclude the possibility that other proteins could interact with FimX. Our results coupled to previous ones, allow us to assess that there is no major structural rearrangement on EAL domain of FimX whether it is in the apo- form or bound to 5′-pGpG or c-di-GMP. Recent results obtained by mass spectrometry on full-length FimX indicate that the binding of c-di-GMP induces long-range conformation changes and that this allosteric regulation could modulate the binding of a partner protein As a conclusion, we bring here the first high-resolution structure of an EAL domain complexed with 5′-pGpG. This is the first time this reaction product of c-di-GMP hydrolysis is trapped within its ligand binding pocket, like a bird in a cage. The analysis of the new network of interactions between the protein, the ligand, a water molecule and the metallic cation support the model of hydrolysis of c-di-GMP by EAL domains proposed previously.Pseudomonas aeruginosa PA01 genomic DNA and cloned into the pET-15b vector (Novagen). The complete FimX was amplified using forward primer 5′-CACAACCATATGGCCATCGAAAAGAAAACCATC and reverse primer 5′-ATACGGGATCCTCATTCGTCTCCCGAGGAGAAGTCGTA using HotStart HiFi Taq DNA polymerase (Qiagen). The PCR product was digested with NdeI and BamHI (New England Biolabs) and cloned into NdeI and BamHI restriction sites. Similarly, GGDEF-EAL domain was amplified using forward primer 5′-GGCGAGCATATGAACGCCGAGCTGGAAGAAAAGCTC and reverse primer 5′-TGCATACTCGAGTCATTCGTCTCCCGAGGAGAAGTC and cloned into NdeI and XhoI sites. EAL domain was amplified with forward primer 5′-GCCGCCCATATGCAGCGCGGCGACGTCATCGCGATC and reverse primer 5′-TGCATACTCGAGTCATTCGTCTCCCGAGGAGAAGTC and cloned into NdeI and XhoI sites.The full-length FimX protein (amino acids 1–691), the GGDEF-EAL domains (amino acids 256–691) and the EAL domain (amino acids 439–691) were amplified by PCR from Escherichia coli BL21 strains were transformed with the recombinant plasmids for protein expression. All the N-terminal his-tagged proteins were purified using the same protocol. Bacteria were grown at 37°C up to OD 0.6. Recombinant protein production was induced by addition of 0.5 mM IPTG. Bacteria were then grown for a further 3 hours at 37°C. Cells were harvested by centrifugation at 4000 g for 20 minutes. The bacteria pellet was re-suspended in lysis buffer  and sonicated for 3 minutes. The sonicate was clarified by centrifugation at 10000 g for 30 minutes. The supernatant was loaded onto a Ni2+-NTA column (Qiagen). The column was extensively washed with lysis buffer and the protein was eluted with NaH2PO4 50 mM, NaCl 300 mM, Imidazole 500 mM. The protein was then purified by size exclusion chromatography on a HiLoad Superdex 200 16/60  equilibrated in Tris-HCl 50 mM, NaCl 300 mM at a flow rate of 1 ml/min. Fractions of interest were analysed on SDS-PAGE and pooled. Proteins were further concentrated on centricon (Amicon).p-nitrophenyl) phosphate (BpNPP) (Sigma-Aldrich) as substrate. BpNPP was diluted in 200 mM NaCl, 50 mM Tris-HCl pH 8.5 and 1 mM MnCl2 to the final concentrations of 1 mM, 5 mM, 10 mM, 15 mM and 20 mM p-nitrophenol) was monitored and quantified at 410 nm at different times. Absorption curves were plotted against time and kinetics parameters extracted from Hanes-Woolf linearization Phosphodiesterase activities of FimX-EAL, FimX GGDEF-EAL and full-length FimX was measured with Bis was used for crystallization. Crystallisation conditions were screened with the JCSG kit (Qiagen) on a CyBIO crystallisation robot. FimX-EAL alone crystallized in Na-Acetate 0.1 M pH 4.5, K2HPO4 0.4 M, NaH2PO4 1.2 M. Crystals of FimX-EAL complex were obtained by vapour diffusion in Hepes 0.1 M pH 7.5, NaH2PO4 0.8 M and K2HPO4 0.8 M. Crystals were cryoprotected with a brief soaking in a solution containing crystallisation buffer and 30% (v/v) glycerol. Diffraction data were collected on Proxima 1 beamline at SOLEIL synchrotron, processed with XDS FimX-EAL (0.5 mM) alone or in equimolar complex with MgCl2O. P. aeruginosa FimX-EAL was dissolved from a concentrated aqueous stock solution by the 0.5 mM ligand NMR solution to a final concentration of 30 uM. 1D proton spectra were recorded using gradient sculpting for water suppression. Reverse NOE-pumping experiments NMR spectra were recorded on a 600 MHz Bruker Avance spectrometer equipped with a TBI probe and X,Y,Z-gradients and samples loaded in a 5 mM Shigemi tubes. The ligands  were dissolved in water to a concentration of 0.5 mM and the pH adjusted to 6.5 by the addition under stirring of small amounts of NaOH 0.1 N. The solutions were then lyophilized and re-suspended in the same volume of DCoordinates and structure factors have been deposited in the Protein Data Bank with accession number 4AG0 (FimX-EAL apo) and 4AFY. (FimX-EAL in complex with 5′-pGpG).Figure S12 1 mM, pH 7.5. (A) Absorbance of the elution profile is recorded at 260 nm and 280 nm. Scattering data corresponding to the first peak (16.68 min) is shown (B) with corresponding Guinier analysis (C). Estimated molecular weight is 33362 Da (expected 30245 Da for FimX EAL monomer)SAXS experiment on FimX EAL in Tris-HCl 50 mM, NaCl 200 mM, MgCl(TIF)Click here for additional data file.Figure S22 1 mM, pH 7.5. (A) Absorbance of the elution profile is recorded at 260 nm and 280 nm. Scattering data corresponding to the first peak (16.738 min) is shown (B) with corresponding Guinier analysis (C). Estimated molecular weight is 28336 Da (expected 30245 Da for FimX EAL monomer). The peak at 19.31 min is free 5′-pGpG.SAXS experiment on FimX EAL mixed with 5′-pGpG (in 100 fold excess) in Tris-HCl 50 mM, NaCl 200 mM, MgCl(TIF)Click here for additional data file.Materials and Methods S1Materials and methods for SAXS experiments.(DOC)Click here for additional data file.