Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab,  Carcinoscorpius rotundicauda, comprises serine protease cascades that are similar to the blood coagulation, melanization and complement systems Serine proteases play an important immunomodulatory role in host-pathogen interactions. Invertebrates lack an adaptive immune system that recognizes and remembers specific pathogens Carcinoscorpius rotundicauda that was infected with Pseudomonas aeruginosa, two isoforms of the non-classical Kazal-type inhibitor, CrSPI, each of 9.3 kDa, were discovered −9 M. Hence subtilisin was used to maximize the exploration of the activity of CrSPI-1. Moreover subtilisin is secreted in large amounts by many bacillus species like Bacillus subtilis. B. subtilis thrives in the natural habitat (estuarine muddy swamp) of the horseshoe crab (C. rotundicauda), although there is no report that B. subtilis is a pathogen of C. rotundicauda.In parallel, a large number of protease inhibitors are also expressed in hemocytes with varying specificities, which target the proteases of different microorganisms Serine protease inhibitors in the plasma have been known to perform dual functions: (i) regulation of the activity of endogenous serine proteases and (ii) inhibition of microbial serine proteases as a host immune defense against invading pathogens The Kazal family is 1 of 18 families of serine protease inhibitors. Since the 1980′s, the structures of several members of this family have been reported Anemonia elastase inhibitor, Crayfish inhibitor Ciona trypsin inhibitor Galleria trypsin inhibitor 1-X2-X3-Cys portion and is cross-linked by two disulfide bridges. Representatives of this group are the Leech Derived Trypsin inhibitor (LDTI-C) Hirudo nipponiaRhodnius prolixusDipetalogaster maximus and Ascidian Trypsin Inhibitor Both domains 1 and 2 of CrSPI-1 share sequence homology with the non-classical group I Kazal-type inhibitors. Other representatives of the non-classical group I Kazal-type inhibitors are the Carcinoscorpius rotundicauda, which thrives in microbiologically-challenging habitat, would exhibit unique antimicrobial properties. Moreover, there are no crystal structures available for a non-classical group I Kazal-type inhibitor, except for the solution structure of the Anemonia elastase inhibitor from the sea anemone Carcinoscorpius rotundicauda Furin), a subtilisin homolog of the host that belongs to the family of Furins or kexins, which are known to be involved in the processing of protein precursors, including many immune proproteins Although several structures have been reported for Kazal-type inhibitors, a structure has not been reported for a protease inhibitor from the hemolymph of an ancient arthropod. The horseshoe crab has been dubbed a “living fossil” with several hundred million years of evolutionary success, indicating that it harbors a formidable antimicrobial system. Therefore, it is conceivable that the CrSPI from the E. coli with the pET32-EkLIC system (Novagen) using the following primers: Forward 5′ ACG GAC GAC GAC AAG ATG TGT CCT CAT ACT TAC AAA 3′ and Reverse 5′ ACG GAG GAG AAG CCC GGT TTA CAA GCA AGC TTC TAG TGG 3′ 600nm of 0.6. Cells were then harvested by centrifugation  and sonicated. The protein was purified using Nickel-NTA affinity beads (Qiagen) with Phosphate Buffered Saline (PBS) at pH 7.4 and 10 mM β-mercaptoethanol (2BME). The protein was eluted in 300 mM imidazole. The thioredoxin tag was cleaved through a 2-h incubation with enterokinase (Sigma). The complex was prepared by mixing CrSPI-1 with subtilisin Carlsberg obtained from Bacillus licheniformis, (Sigma) in an approximate molar ratio of 1∶2 (inhibitor to enzyme) and incubated for 1 h at 37°C. The complex was purified using a Superdex-75 gel filtration column, and fractions were pooled and concentrated up to 20 mg/ml. Crystallization screens were performed using the hanging drop vapor diffusion method with Hampton Research Crystal Screen kits I and II and JB Screens  at room temperature. The initial crystallization conditions were further optimized using a grid screen by varying the concentration of the precipitant. Plate-like diffraction quality crystals were obtained from 11% (w/v) Polyethylene Glycol 20000 in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) at pH 6.5. The crystallization solution was supplemented with 25% glycerol, which acts as a cryo-protectant. A complete dataset was collected in the X29-A synchrotron beamline at Brookhaven National Laboratories, USA. The data collection and refinement statistics are provided in The CrSPI-1 gene was cloned and expressed in The structure of the recombinant CrSPI-1: subtilisin complex was solved by the molecular replacement method with Molrep ITC studies were performed using the MicroCal VP-ITC and MicroCal ITC-200 instruments. CrSPI-1 at a concentration of 0.2 mM in PBS at pH 7.4 and 10 mM BME, was titrated into a solution of 0.012 mM subtilisin in PBS at pH 7.4 and 10 mM BME in the sample cell with injections of 2 µL (ITC-200) or 10 µL (VP-ITC). Additionally, the VCTEEY peptide, at a concentration of 11 mM in PBS at pH 7.4 and 10 mM BME, was titrated into a 0.063 mM subtilisin solution in PBS at pH 7.4 and 10 mM BME in the sample cell with injections of 1 µL. This peptide was designed based on the reactive site loop sequence of CrSPI-1 that is in the proximity of the active site of subtilisin in the CrSPI-1: subtilisin complex in the crystal structure. The VCTEEY peptide, which contains the RSL residues from P4 to P2′ of CrSPI-1 domain 2, was purchased from Sigma-Aldrich. All experiments were performed at 37°C. Samples were degassed prior to use. Due to dilution across the needle during equilibration, the initial peak was discarded. Data analysis was performed using the MicroCal Origin software. For the rCrSPI-1 experiments, a model considering an interaction with a binding stoichiometric ratio of 1∶2 CrSPI-1: subtilisin  in terms of the overall association constants or the site-specific intrinsic microscopic association constants was employed free = 0.24) at 2.6 Å resolution with good stereochemical parameters  structure and structures of subtilisin complexed with small ligands The CrSPI-1 molecule comprises two domains: domain 1 from Cys1 to Glu40 and domain 2 from Leu41 to Leu83. Both domains adopted similar secondary structures (domain 1 β1↑β2↓α1β3↑ and domain 2 β4↑β5↓α2). The presence of a central α-helix, α1 (Glu18–Ala24) in domain 1 and α2 (Arg63-Ser68) in domain 2, and an antiparallel β-sheet in each domain are characteristic of the classical Kazal-type inhibitors. However, CrSPI-1 also showed features of a non-classical Kazal-type serine protease inhibitor in that it harbored an unusual pattern of conserved cysteines. There were two intra-domain disulfide bridges in domain 1 (Cys1-Cys19 and Cys8-Cys35) and three in domain 2  . Using aA search for topologically similar proteins within the PDB database was performed with the DALI program et al.The comparison of CrSPI-1 domains 1 and 2 revealed that the core regions superimposed with an rmsd of 3.9 Å for 24 Cα atoms . The seqRhodnius prolixus, was shown to inhibit thrombin at a 1∶1 ratio by binding of one domain at the active site and the other domain at the exosite The CrSPI-1: subtilisin complex is a heterotrimer in solution, which was confirmed by gel filtration, non-reducing SDS-PAGE γ of the catalytic Ser220 (2.63 Å). The carbonyl O atom of the P1 His3 interacted with the backbone NH group of the active site Ser220 and Nγ2 of Asn154 of subtilisin (The reactive site loop (RSL) of domain 1 of CrSPI-1 (Cys1 to Lys6) binds at the active site region of subtilisin from P3 (Cys1) to P3′ (Lys6) in a substrate-like manner , panel 1btilisin . In Kazabtilisin , panel 1In domain 2 of CrSPI-1, Glu49 takes the position of His3 in the P1 pocket of domain 1 and makes 8 (73%) of the 11 total hydrogen bonding contacts with subtilisin. The remaining hydrogen bonding contacts with subtilisin are from P1′ Glu49, P2′ Tyr50 and P4 Val45 of domain 2 . SimilarThe conformations adopted by the reactive site loops (RSLs) of both domains of rCrSPI were similar and superimposable with an rmsd of 0.80 Å for the residues from positions P3 to P3′ . The RSLThe backbone torsion angles (ψ/ϕ angles) of the RSLs of CrSPI-1 were similar to each other and to other protease inhibitors, such as OMTKY3 and Eglin-C complexed with subtilisin . In addiSuperimposition of the active site region of the subtilisin complexes of OMTKY3 and Eglin C with the CrSPI-1: subtilisin complex revealed that different inhibitor loop sequences can be accommodated within the substrate binding site of subtilisin with minimal side chain rearrangement . Furtherδ2 of Asn18 with the main chain carbonyl atoms of Pro2 and Thr4 at the P2 and P1′ positions of the RSL. Similarly, in domain 2, the interactions through the side chains of Asn62, Thr47 and Glu49 maintain the rigidity of the RSL. The interactions between Thr47 and Glu49 are similar to the interactions of P2-P1′ of domain 1. In order to reduce the entropic cost of binding, the RSLs of Kazal-type inhibitors and of CrSPI-1 in particular are firmly held in a preferred conformation that is complementary to the substrate-binding site of specific proteases. A rigid conformation of the RSL is thought to prevent proteolytic cleavage of the inhibitor upon interaction with proteases In addition to the S-S bridges, there are three important internal hydrogen bonding contacts that maintain the rigidity of the RSLs in CrSPI-1. These contacts in domain 1 are (1) the P2-P1′ hydrogen bond between the carbonyl oxygen of Pro2 and amide nitrogen of Thr4 of the reactive site loop, (2) the hydrogen bond between Asn18 and Phe21, and (3) the interaction of Na) of 2×103 M−1 (Kd = 500 µM)  derived from the RSL region of domain 2 of CrSPI-1 using Isothermal Titration Calorimetric (ITC) experiments . Titrati 500 µM) . This inPhytophthora infestans2 and 530 Å2 of buried surface area, respectively, which accounts for approximately 76% and 90% of the binding interfaces with subtilisin, respectively. Thus, the RSL of domain 2 contributes a greater buried area compared to the RSL of domain 1. Notably, domain 2 inhibits subtilisin with a Ki of 2.6 nM Kazal-type serine protease inhibitors are single or multi-domain proteins with domains that usually have different specificities towards a particular protease ex-vivo inhibition assay Previously we have shown the inhibition of CrFurin by CrSPI-1 in an Bacillus species, Furin is considered as a eukaryotic version of subtilisin, which is ubiquitously expressed Furin is a subtilisin-related serine protease, and a member of the proprotein convertase family. Although, subtilisin is obtained from bacteria belonging to the The preference of CrSPI-1 domain 2 for the microbial protease subtilisin suggests that this isoform targets serine proteases of invading microbes. In fact, subtilisin is the virulence factor used by the pathogen to gain entry into host cells during an infection. Besides direct suppression of the microbial proteases, CrSPI-1 has been shown to target the host's endogenous proteases like the CrFurin. This is supported by the observation that in response to an infection, a dynamic reciprocal expression profile of the CrSPI-1 and CrFurin transcripts was observed Figure S1Alignment of amino acid sequences of non-classical group I Kazal-type inhibitors AEI, CrSPI-1 domain I, domain II and a selected classical Kazal-type inhibitor OMSVP3. The sequences were aligned using CLUSTAL-W. The reactive site is denoted with an arrow. Disulfide bonds are linked as follows: α-β, II-IV, and III-VI for the non classical group I inhibitors and I-V, II-IV, and III-VI for the classical inhibitors. In nonclassical group II inhibitor family, there is an additional disulphide bridge between α and β half cystines.(TIF)Click here for additional data file.Figure S2Conformations of the reactive site loop (RSL). Superimposition of the reactive site loops of domain-1 (magenta), domain-2 (yellow), Eglin C (gray) and OMTKY3 (cyan). The RSLs are shown in stick representation whereas the substrate binding site of subtilisin is shown in surface representation. These figures were generated by using PyMol.(TIF)Click here for additional data file.Figure S36 M−1 and 1.7x104 M−1 were obtained from non-linear regression analysis, corresponding to dissociation constants of 0.42 and 59 μM, respectively.Isothermal Titration Calorimetric (ITC) curve for rCrSPI-1 titrated against subtilisin at 37°C. Each peak represents the injection of rCrSPI-1 0.2 mM into the ITC cell containing subtilisin 0.012 mM, in buffer PBS pH 7.4, 10 mM BME. A sequence of 18 injections, each injection consisting of 2 μL of ligand solution, was performed. The experimental data were fitted considering a model in which CrSPI-1 binds two Subtilisin molecules, either employing a general model based on the overall association parameters or considering two non-identical and independent binding sites in CrSPI-1 (32). Binding association constants of 2.4x10(TIF)Click here for additional data file.Figure S4Cα trace for the heterotrimer Furin-CrSPI-Subtilisin complex model. Furin and subtilisin share a sequence identity of 23%. The Furin:CrSPI-1 complex model was generated by superimposing domain-1 CrSPI-1: subtilisin complex onto the structure of Furin (pdb code 1p8j), which yielded an rmsd of 2Å for 268 Cα out of 274 Cα atoms of subtilisin. The Furin-CrSPI-1-Subtilisin heterotrimer complex was generated using the modeled Furin-CrSPI-1-domain-1 and subtilisin-CrSPI-1-domain-2 complex crystal structure.(TIF)Click here for additional data file.Figure S5Surface representation for Furin and Subtilisin, and backbone trace representation for CrSPI-1 of the heterotrimer model.(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.Table S3(DOC)Click here for additional data file.


Subjective duration is strongly influenced by repetition and novelty, such that an oddball stimulus in a stream of repeated stimuli appears to last longer in duration in comparison. We hypothesize that this duration illusion, called the temporal oddball effect, is a result of the difference in expectation between the oddball and the repeated stimuli. Specifically, we conjecture that the repeated stimuli contract in duration as a result of increased predictability; these duration contractions, we suggest, result from decreased neural response amplitude with repetition, known as repetition suppression.Participants viewed trials consisting of lines presented at a particular orientation (standard stimuli) followed by a line presented at a different orientation . We found that the size of the oddball effect correlates with the number of repetitions of the standard stimulus as well as the amount of deviance from the oddball stimulus; both of these results are consistent with a repetition suppression hypothesis. Further, we find that the temporal oddball effect is sensitive to experimental context – that is, the size of the oddball effect for a particular experimental trial is influenced by the range of duration distortions seen in preceding trials.Our data suggest that the repetition-related duration contractions causing the oddball effect are a result of neural repetition suppression. More generally, subjective duration may reflect the prediction error associated with a stimulus and, consequently, the efficiency of encoding that stimulus. Additionally, we emphasize that experimental context effects need to be taken into consideration when designing duration-related tasks. The perceived duration of a stimulus is modulated by repetition and novelty. Consequently, the first stimulus in a stream of repetitions appears expanded in comparison to successive ones In this paper, we test the above hypothesis by focusing on some characteristics of repetition suppression and probing for their perceptual correlates in the oddball effect. First, repetition suppression has been shown to increase with increasing number of presentations, until it hits saturation by around 6–8 presentations All participants were between 18–45 years of age and had normal or corrected-to-normal vision. Research was approved and all participants were consented in writing according to the procedures of the Institutional Review Board at Baylor College of Medicine.Stimuli consisted of a blue line  that was presented at the center of the screen against a black background. On each trial, a blue line (of a fixed orientation) repeatedly appeared for 500 ms with an interstimulus interval of 300 ms. After a certain number of repetitions, an oddball line – a line that was rotated at an certain angle from the standard line – would appear followed by more standard lines. The oddball line was presented for different physical durations ranging from 300 to 700 ms (in steps of 50 ms). On each trial, 7 lines, including the oddball, appeared one after the other. At the end of the trial, participants were required to respond to whether the oddball stimulus was longer or shorter in duration than the standard lines. The next trial began 1000 ms following their response.In this experiment, the oddball line, which was always rotated 45° away from the standard, could appear anywhere from the 2nd through the 6th position in a serially-presented visual train . The ori11 participants ran the experiment. Psychometric curves were fit to each participant's data to quantify the point of subjective equality (PSE) of the standard and oddball stimuli, i.e. the point at which the participant reported the oddball was longer than the standard 50% of the time. From this PSE, the duration distortion factor (DDF) was calculated as the ratio of the standard duration to the PSE for the oddball 4,40 = 18.496; p<2.86e-06, corrected for sphericity using the Huynh-Feldt correction). Further, we tested a linear mixed model in R using the lmer function 1,18 = 56.9; p<2e-16). This effect of number of repetitions is reminiscent of repetition suppression As is evident from Neural activity suppression can occur not only as a result of repetition, but also when a non-repeated stimulus is predictable To address whether the degree to which the deviance of the oddball from the standard stimulus modulates the oddball effect, we varied the difference in angle between the oddball and the standard lines . This ex4,28 = 21.77; p<7.78e-07 corrected for sphericity, and F4,60 = 10.05; p<0.00078 corrected for sphericity, one-way repeated measures ANOVA). The size of the oddball effect increased monotonically with the degree of novelty from 0 to 20 degrees . This finding is consistent with those of Schindel et al (2011), who also demonstrated that oddball effects scaled with the angular discrepancy between repeated and oddball stimuli. Further, one-sampled t-tests against mean 0 on the slope of the increase from 10°–50° was statistically significant , but not from 50°–90° , indicating that the effect of novelty appears to saturate by about 50° of difference  even though the stimuli are identical in both cases. This difference is suggestive of an experimental context effect on duration distortion. In light of this, we next asked whether the temporal context of the oddball (its position in the sequence) might also influence the duration distortion. To address this question, we varied the position of the oddball in the visual train as shown in th position. In all cases, the oddball was the final stimulus in the visual train. We predicted that the size of the oddball effect at the 7th position would differ between the blocks as a result of a different temporal context.From 21 = 2.97, p<0.008). There was no statistical difference between the DDF in the block in which the oddball always appeared at the 7th position relative to the other two blocks .Our data indicate that both the number of repetitions of the standard stimuli preceding the oddball and the relative novelty of the oddball play a considerable role in determining the size of the oddball duration effect. The novelty findings  are consWhile we have explored the immediate effects of repetition here, repetition also involves long-term, familiarity-like effects consequence of its violation Other researchers have suggested attentional explanations for the oddball effect It is worth noting that participants were instructed to judge the duration of the oddball with respect to the stimuli that come before it; they may, therefore, be doing one of two things: 1) comparing the duration of the oddball to the standard stimulus immediately preceding it, or 2) comparing the duration of the oddball to the average duration of all the standard stimuli preceding it in the trial. These comparison strategies may vary from individual to individual. Regardless of how the participant is carrying out the comparison process, our interpretation of the data remains the same. The size of the oddball effect for an individual may vary based on the strategy employed, but the effect of number of repetitions or of the degree of novelty should be present in qualitatively the same fashion for all participants.We also found that the experimental context—that is, the set of possible oddball positions within the experiment—strongly influences the oddball effect. A similar effect was seen in Tse et al th position in the 4/5/6/7 condition, and minimal in the 7/8/9/10 condition. Our neural response amplitude hypothesis Why is subjective duration affected by experimental context? For Experiment 2, one framework for understanding this is found in Helson's adaptation level theory, in which stimulus judgments are influenced by the range of previously encountered stimuli In summary, our results provide a prediction to be tested using electrophysiology: the degree of duration distortion of an oddball stimulus mirrors neural response magnitudes, which is itself modified by predictive coding and experimental context.


Uca vomeris defend their burrows against intruders and it seems they use a burrow-centered reference coordinate. The crabs respond when an intruder approaches the burrow within some distance of the burrow. There are several possible cues to estimate the distance between the intruder and the burrow entrance by visual information such as disparity, optical flow, image size and the retinal elevation. However, experiments show only specific visual information is related to the burrow surveillance [A fiddler crab can estimate how close an intruder is from its burrow . It is beillance .The retinal position, azimuth and elevation angle of the intruder in view is related to the distance between the intruder and the observer, following the geometric function. The former parameters can determine the intruder-crab distance depending on the crab-burrow distance. Fiddler crabs on leaving home accumulate the distance and direction of the exploring path can determine the homing distance and direction from the current position to the burrow . To deteThere has been a suggestion that the burrow surveillance in fiddler crabs may be connected with directionally sensitive neurons and distance neurons . Our exp


The current version also includes a pipeline for the discovery and annotation of novel domain–motif interactions. For every interaction, 3did identifies and groups different binding modes by clustering similar interfaces into ‘interaction topologies’. By maintaining a constantly updated collection of domain-based structural interaction templates, 3did is a reference source of information for the structural characterization of protein interaction networks. 3did is updated every 6 months.The database of 3D interacting domains (3did, available online for browsing and bulk download at  Proteins are key players in virtually all events that take place within and between cells. However, they seldom act alone and it is their complex interrelationships that will ultimately determine the behavior of a biological system. For this reason, large efforts have been devoted to unveiling the complex network of interactions between proteins underlying biological processes, producing large interactomes for several organisms, including human . High-thSeveral efforts over the last years aimed at mining the data in the PDB to provide a comprehensive structural characterization of protein interaction networks . While tSeveral bioinformatics studies have attempted to define and classify domain interactions, both DDIs  and DMIsThe database of 3D interacting domains (3did) is a collection of 3D structures of domain-based interactions, both DDIs and DMIs, based on domain definitions from Pfam , ensurin2 on average  or between two different chains (interchain). We require at least five estimated contacts  in ordebased on ,27]. Forbased on .The current version of 3did contains 258 079 structural instances of DDIs of which 68 861 are intrachain and 189 218 are interchain. These correspond to 8328 unique domain–domain pairs (1190 with only intrachain instances and 5747 with only interchain instances while 1391 have both intra and interchain instances). With respect to the last version of 3did (2011) we observed an impressive growth of 62% in the number of DDI structures corresponding to 39.5% more domain–domain distinct (i.e. nonredundant) pairs, reflecting the constantly increasing rate of growing of the PDB and Pfam . Alternatively it is possible, through the ‘browse’ tab, to browse all domains and motifs present in 3did or to explore a GO tree and retrieve all the domains associated to any GO term , a motif name, a structure (by PDB ID) or any term from the Gene Ontology . The ass GO term C.http://www.jmol.org/) the first structure  in which the interaction has been identified. Different interfaces, involving different residues, may be identified for each interaction. Those different interfaces, or topologies, are also listed in the interaction page, with the residues involved. In addition, the list of structures in which the interaction has been found is provided: each structure can be displayed in Jmol by clicking the associated ‘View’ button. Finally, the PDB view allows displaying all domains and motifs identified in a specific PDB structure as well as their interactions. The DDIs and DMIs are represented as a network in CytoscapeWeb and the structure is displayed in Jmol. In addition, the domain architecture of each chain in the PDB file is listed, as well as the interactions in which each domain of the respective chain is involved ].Funding for open access charge: Conflict of interest statement. None declared.


Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication. Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of  LD50 per mg [2+-endopeptidases [The seven Botulinum neurotoxins (BoNT/A-/G) are bacterial metalloproteases that act in the cytosol of cholinergic nerve terminals. They cleave core proteins of the neuroexocytosis apparatus causing a long-lasting inhibition of neurotransmitter release .BoNTs ar0 per mg .The discptidases  has led ptidases .Protein-protein interactions play a central role in many cellular functions, and as whole-genome data accumulates, computational methods for predicting these interactions become increasingly important. Computational methods have proven to be a useful first step for rapid genome-wide identification of putative protein structure and function. In the genomic era, one of the most interesting and important challenges is to understand protein interactions on a large-scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. Protein domain interactions are essential to the functioning of individual cells and whole organisms by acting in several ways: domain-domain interactions in multi-domain polypeptide chains, inter-chain protein interactions in obligate complexes such as multimers or oligomers and in transient complexes between proteins that can also exist independently. Therefore, it is not surprising that protein interactions have been extensively investigated using various methods. de novo design [The parallel two-stranded α-helical coiled-coil is the most frequently encountered subunit oligomerization motif in proteins. The simplicity and regularity of this motif have made it an attractive system to explore some of the fundamentals of protein folding and stability and in testing the principles of o design . Despiteo design . In this study, we have examined the protein domains of seven serotypes derived from published amino acid sequence of BoNTs serotypes. All BoNT serotypes have common protein domains present except BoNT/B and BoNT/G. Our analysis provides evidence that BoNT/B and BoNT/G do not have coiled-coil domains and they appeared to be derived from a common ancestorThe crystallographic structure of BoNT/A and BoNT/B molecules consists of three structural domains matching to three functional domains, namely catalytic, translocation and binding . These ti.e., the coiled-coil motif), can be often recognized from the primary structure of the protein. One coiled-coil motif (4-3 hydrophobic repeats) is a heptads repeat of amino acids from A to G so that A and D are hydrophobic and E and G predominantly polar. The coiled-coil structure is used by nature to stabilize α-helices in proteins. This is possible through a very efficient burial of hydrophobic side chains in proteins so that for the most part, they are not accessed by polar water molecules. Because of this structure, the helices are quite stable. Many structural proteins both inside and outside of cells  have a coiled-coil domain. There seem to be basic, but essential, features that are common to coiled-coil peptides. First, the overall secondary structure is alpha helical and secondly, the hydrophobic residues are arranged on one side of the helices. The typical positioning of the hydrophobic residues in coiled-coils,  binds to the surface of target nerve cells and the N-terminal part (HN) is involved in the translocation the light chain across the membrane [The existence of a coiled-coil structure in close proximity to the translocation domain of BoNT/A, however, raises important questions concerning its exact function for blocking the synaptic transmission. Except for BoNT/B and BoNT/G, all other serotypes contain coiled-coil regions within the membrane . The twomembrane . These tmembrane ,16. CD amembrane .N-terminus of heavy chain that wraps around the catalytic domain might act as a chaperone assisting in light chain unfolding, channel transit, and refolding [N-terminal part which is involved in the translocation,in BoNT/A differs from BoNT/B, especially at residues 481-532 that wrap around the catalytic domain, we think that these differences should be taken into account inferring any common translocation mechanism of all BoNTs. However, absence of coiled-coil in BoNT/B and BoNT/G , bind to a pocket at the tip of the ing site ,23. In aer BoNTs . C-terminal domain of the C-terminal fragment of the heavy chain, we believe that N-terminal part which involve the translocation of the light chain across the membrane could be playing a different role in these two serotypes. Note that our computational predictions lack biochemical evidences and do not reach yes-or-no decisions based on a threshold value. Rather, it yields a set of probabilities that presumably reflect the coiled-coil forming potential in BoNTs published sequences. This means that even at high probabilities there will be (and should be) sequences that in fact do not form a coiled coil, though they may have the potential to do so in a different context. Moreover, the program we used to predict coiled coils cannot predict on buried structures. While there appears to be a similarity in the receptor binding property of BoNT/B and BoNT/G which is mediated by N-terminal part) causes conformational changes there by releasing the L-chain after a disulfide reduction. Under physiological conditions, the L-chain is believed to separate from the H-chain at the endosomal membrane, and the L-chain is only active after reduction of the disulfide bond. To release the L-chain, we believe that it is possible that self oligomerization of the H-chain  protein sequences from seven serotypes were obtained from NCBI and analyzed for sequence comparison using the GCG program . (ii) multiple sequence alignment was performed using CLUSTAL V based on algorithm of Higgins . The domC. botulinum neurotoxins was performed using CLUSTALX 1.81 for multiple sequence alignment and Tree-View for neighbor joining tree construction with maximum bootstrapping option [Phylogenetic analysis of seven serotypes of g option . A majorThese results indicate that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomarization of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication. A detailed understanding of the architecture, catalysis, and recognition properties of these domains will also help to reveal how the toxins achieve functional diversity.


Selective autophagy is a process whereby specific targeted cargo proteins, aggregates, or organelles are sequestered into double-membrane-bound phagophores before fusion with the lysosome for protein degradation. It has been demonstrated that the microtubule network is important for the formation and movement of autophagosomes. Nbr1 is a selective cargo receptor that through its interaction with LC3 recruits ubiquitinated proteins for autophagic degradation. This study demonstrates an interaction between the evolutionarily conserved FW domain of Nbr1 with the microtubule-associated protein MAP1B. Upon autophagy induction, MAP1B localisation is focused into discrete vesicles with Nbr1. This colocalisation is dependent upon an intact microtubule network as depolymerisation by nocodazole treatment abolishes starvation-induced MAP1B recruitment to these vesicles. MAP1B is not recruited to autophagosomes for protein degradation as blockage of lysosomal acidification does not result in significant increased MAP1B protein levels. However, the protein levels of phosphorylated MAP1B are significantly increased upon blockage of autophagic degradation. This is the first evidence that links the ubiquitin receptor Nbr1, which shuttles ubiquitinated proteins to be degraded by autophagy, to the microtubule network. Cellular turnover of damaged and misfolded proteins is mediated by two main degradation pathways; macroautophagy (hereafter referred to as autophagy) and the ubiquitin proteasome system (UPS). The UPS targets soluble, cytosolic proteins to the proteasome where they are degraded. Proteins targeted for degradation are covalently modified by the small, highly conserved, ubiquitously expressed protein ubiquitin. Ubiquitin can form chains at all seven lysine residues and typically, chains of four or more ubiquitin molecules are required for the targeting of proteins to the proteasome . HoweverAutophagy requires the coordinated action of 35 to date autophagy-related genes (ATG) that mediate the formation of the double-membrane bound autophagosome which encloses a portion of the cytoplasm and delivers it to the lysosome . There aRecent evidence demonstrates that autophagy can be a selective process, whereby single proteins and cellular structures such as aggregates and organelles can be specifically targeted to autophagosomes , 16, butIn this paper, we identify NBR1 as an interaction partner of the microtubule-associated protein MAP1B. This occurs via the evolutionarily conserved FW domain. We show that whilst MAP1B is not itself a substrate for autophagosomal protein degradation, the phosphorylated form of MAP1B is stabilised by lysosomal inhibition. We propose that this interaction provides a mechanism by which NBR1 is targeted to the microtubule network to promote degradation of proteins via the autophagosome.BioEdit was used to curate sequences and compile alignments. BLAST was used on various databases to identify FW-like sequences from animal, plant, fungal, protist, bacterial, and metagenome sequences. Phyre was used for structural predictions.β-actin (Abcam), and monoclonal anti-His (Novagen).For western blot analysis and immunofluorescence the following antibodies were used: polyclonal anti-myc , monoclonal anti-HA (Roche), monoclonal anti-myc , and polyclonal anti-MAP1B-HC  (Clontech). Full length Nbr1 was cloned into pHM6 (Roche) and MAP1B aa2216- 2464 was cloned into pcDNA3.1 (Invitrogen) for the coimmunoprecipitation assay. Nbr1 aa346-498 was cloned into pGEX2T  for the GST-binding assay and MAP1B aa2227-2464 was cloned into PET6H (a modified version of pET11d-Novagen) for the recombinant binding assay.Yeast strain Y187 was transformed with the Nbr1 bait construct and mated with a pretransformed (yeast strain AH109) mouse neonatal calvarial cDNA library kindly supplied by Prof. Ikramuddin Aukhil, University of Florida. Resulting colonies were screened by HIS3 reporter gene activity, replated three times and inserts were sequenced. Y187 transformed with pGBKT7 Nbr1 aa346-498 was mated with yeast strain AH109 expressing the library MAP1B clone pGADT7 MAP1B aa2238-2465 and plated onto SD medium lacking leucine, tryptophan, histidine and adenine and were cultured at 30°C to verify the interaction.COS7 cells were cotransfected with HA-Nbr1 and MAP1B-myc and after 48 hours, lysed in IP buffer ), and cell lysates incubated with rabbit polyclonal anti-myc antibody overnight at 4°C. Protein A beads (Millipore) were then added to the lysates for a further 2 hours, beads were then washed three times in IP buffer. Proteins retained on the beads were separated by SDS-PAGE and transferred onto a nitrocellulose membrane following standard procedures. Blots were probed with mouse monoclonal anti-myc and rat monoclonal anti-HA antibodies and subsequently with a secondary antibody . Detection was performed by ECL .6-MAP1B aa2227-2464 were lysed in lysis buffer ). The sample was sonicated, centrifuged, and the supernatant was incubated with Ni Sepharose 6 fast flow beads (Amersham Biosciences) for 2 hours at 4°C. Beads were then washed in low Imidazole elution buffer ) and bound proteins eluted from the beads using high Imidazole elution buffer ). The resulting purified His-tagged protein was dialysed into 50 mM Tris pH 7.5, 150 mM NaCl and used in the GST pull-down assay.Nbr1 aa346-498 fused to GST and GST alone were expressed in Bl21(DE3) bacterial cells and proteins purified by glutathione affinity chromatography as previously described . MAP1B a6-MAP1B aa2227-2464 for 2 hours at 4°C. Following incubation, beads were washed three times in IP wash buffer , and proteins retained on the beads were separated by SDS-PAGE and analysed by western blotting as described above, using the anti-His tag monoclonal antibody.COS7 cells were transfected with a MAP1B-myc construct and after 48 hours expression, lysed in IP buffer (as above), and lysates incubated with beads coupled with either GST-Nbr1 aa346-498 or GST alone for 2 hours at 4°C. Following incubation, beads were washed three times in IP wash buffer ) and proteins retained on the beads were analysed by western blotting as described above, using the monoclonal anti-myc antibody, 9E10. Alternatively purified GST or GST-Nbr1 aa346-498 attached to glutathione agarose beads (Sigma) was incubated in IP buffer ) with purified HisμL IP buffer for pull-down and coimmunoprecipitation assays. For immunostaining PC12 cells were cultured on coverslips in DMEM/10% FCS, treated with DMSO or Bafilomycin A1 (Sigma) for 4 or 8 hours, or starved in Hanks-Balanced Salt Solution (Sigma) for 4 hours or treated with 5 μg/mL nocodazole (Sigma) for 30 minutes before or after 2 hours starvation and fixed in 4% paraformaldehyde/PBS for 10 minutes. Cells were then permeabilised in 0.1% Triton X100/PBS and incubated consecutively with primary and secondary antibodies (Dako) for one hour each prior to mounting. Cells were imaged using a Zeiss LSM 510 confocal microscope in sequential scanning mode with a Plan-Apochromat 63 x/1.4 Oil DIC objective. Quantification of MAP1B/Nbr1 colocalisation was performed using Zeiss ZEN2010 software, data represent mean ± SEM of 22 images.COS7 cells were cultured in DMEM/10% FCS by standard protocols and transfected using Fugene 6 (Roche). Cells were lysed 48 hours later in 200 We used BLAST-based searches to acquire NBR1-related sequences from multiple available genomic and transcriptomic sources across a broad range of eukaryotes. These identified a region of pronounced conservation of 105 amino acids (residues 374–478 of human NBR1) which is recognisable in the single NBR1 orthologue found in most eukaryotes but is absent from p62. This novel domain has been named the NBR1 domain  and FW dSaccharomyces cerevisiae) and some single-celled eukaryotes (such as Dictyostelium discoideum). In each case the NBR1-like molecule possessed an N-terminal PB1 domain, one  or more (fungi) ZZ domains, an FW domain, and a C-terminal UBA domain , comprising a universally conserved N-terminal l region .γ-proteobacteria, chloroflexi, actinobacteria, and several unclassified metagenomes). In several of the bacterial proteins , the FW domain appears immediately C-terminal to a robustly predicted “helix-turn-helix” DNA-binding motif of the XRE family. We call these XRE-FW proteins. XRE domains tend to appear either alone or with multimerisation domains . This juxtaposition raises the possibility that the FW domain might mediate homo- and/or heterodimerisation or could bind a small signalling molecule. Some other eubacterial FW proteins consist solely of two tandem FW domains and little else , while others contain transmembrane domains . This structural diversity suggests that the FW domain has a generically useful function that has been exploited in many ways.Intriguingly, FW domains were also found in a wide range of eubacteria. The eubacterial FW-containing proteins are strikingly diverse in domain structure, with the only common theme being that the FW domain tends to be very close to the C-terminus. Although most eubacterial genomes do not encode an FW domain-containing protein, we find that it is broadly distributed across eubacterial clades  argues against multiple eukaryote-to-prokaryote horizontal gene transfer events and suggests that the FW domain may be ancient, predating the split between eukaryotic and eubacterial domains.To identify novel protein interactors of the highly conserved FW domain of Nbr1 and therefore elucidate a function, we performed a yeast-2-hybrid screen with the FW domain of Nbr1 as bait. A neonatal calvarial cDNA library was screened and the light chain of the microtubule-associated protein 1B (MAP1B-LC1) was identified as an interaction partner of Nbr1. This interaction was verified by a directed yeast two-hybrid assay by retransforming the isolated prey vector encoding the partial MAP1B-LC1 sequence aa2238-2465) into yeast strain AH109 and mating it with yeast strain Y187 that was expressing the FW domain of Nbr1 . MAP1B i-2465 intin vivo, we performed a coimmunoprecipitation experiment using COS7 cells transiently transfected with HA-Nbr1 and MAP1B-LC1-myc constructs. Using an anti-myc antibody for immunoprecipitation of MAP1B-LC1-myc, we found that HA-Nbr1 did coimmunoprecipitate with MAP1B-LC1-myc .To determine whether Nbr1 forms a complex with MAP1B-LC1 -LC1-myc  confirmiThe yeast-2-hybrid data suggested that the FW domain of Nbr1 interacts directly with MAP1B-LC1. To more rigorously test this hypothesis, we performed GST pull-down assays using extracts from COS7 cells overexpressing MAP1B-LC1-myc and a GST fusion of the FW domain of Nbr1 and GST alone. Indeed, the FW domain of Nbr1 interacted with MAP1B-LC1 whilst GST alone did not .6-MAP1B-LC1 was purified and incubated with the GST fusions of the FW domain of Nbr1 or GST alone. This demonstrated that the FW domain of Nbr1 interacts directly with the light chain of MAP1B  and resulted in loss of the punctate colocalisation of MAP1B and Nbr1 but intact Nbr1 vesicles were retained  [The high evolutionary conservation of the FW domain within Nbr1 homologues implicates it to have a critical role in Nbr1 function. The predicted secondary structure of the FW domain that consists of two three 2 (NPC2) , 54 suggIn summary, we present the first evidence linking the autophagic receptor protein Nbr1 and the microtubule network via a direct interaction of the evolutionary-conserved FW domain of Nbr1 with MAP1B. Nbr1 is a ubiquitously expressed protein that has been implicated in several diseases , 22, 23,


Ligands for ErbB1-4 receptor tyrosine kinases, such as epidermal growth factor (EGF) and neuregulins, regulate brain development and function. Thus, abnormalities in their signaling are implicated in the etiology or pathology of schizophrenia and Parkinson's disease. Among the ErbB receptors, ErbB1, and ErbB4 are expressed in dopamine and GABA neurons, while ErbB1, 2, and/or 3 are mainly present in oligodendrocytes, astrocytes, and their precursors. Thus, deficits in ErbB signaling might contribute to the neurological and psychiatric diseases stemming from these cell types. By incorporating the latest cancer molecular biology as well as our recent progress, we discuss signal cross talk between the ErbB1-4 subunits and their neurobiological functions in each cell type. The potential contribution of virus-derived cytokines (virokines) that mimic EGF and neuregulin-1 in brain diseases are also discussed. C. elegans  and its derivatives. The ErbB family consists of four members, ErbB1, B2, B3, and B4, that were originally identified in vertebrates  and share significant structural homology with members of the ErbB family  was first elucidated among ErbBs. The oncogene Ligands for ErbB receptors can be classified into two groups: the EGF family and the NRG family Falls, . The EGFerbB genes or their products are subjected to alternative splicing or proteolytic processing, resulting in truncated isoforms lacking the kinase domain or ligand-binding domain. These truncated isoforms function as an enhancer of tumorigenesis, a receptor decoy or a transcription factor , which lacks extracellular domain of EGFR - protein kinase C (PKC), Ras- mitogen-activated protein kinase (MAPK), PI3K-Akt, and janus kinase 2 (JAK2)-STAT3 pathways  are highly enriched with ErbB1  as well as isoforms with truncations in the intracellular domain  in neurons  in dopamine neurons  in the prefrontal cortex and hippocampus of patients  is encoded by the genome of vaccinia virus in the poxvirus family and has an amino acid sequence homologous to EGF Figure . VGF is In addition to soluble virokines, virus-derived effectors can affect intracellular ErbB signaling. E5 is one of the early gene products of human papillomaviruses. E5 protein is also a membrane-anchored molecule, like the EGF precursor, but lacks the EGF-like domain. For example, the E5 protein of human papillomavirus type 16(HPV-16) associates with the ATPase motif of the ErbB1 tyrosine kinase domain, attenuate its interaction with Cbl, and inhibits the internalization and degradation of ErbB1 Figure . In addierbB gene products are present in certain neurons or glia and not the real structures of particular ErbB isoforms in various brain regions. In this context, a more elaborate analysis may be required to accurately discuss their functions in the nervous system.Studies in cancer biology clearly indicate the pathologic powers of abnormal ErbB signaling and its contribution to oncogenesis, asthma, injury repair, and rheumatoid arthritis , is poorly understood even though those are highly expressed in the brain (Kinugasa et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs. Escherichia coli this route is linear and NAG is made from acetyl CoA (AcCoA) and glutamate by NAG synthase (NAGS). However, many other organisms recycle the acetyl group by transacetylation from N-acetylornithine to glutamate  family 3β8α4 sandwich with a mostly parallel central β8-sheet, which is split into N- and C-lobes. All the NAGKs characterized structurally so far Because of the paramount regulatory role of NAGK, this enzyme has been the focal point of studies which have revealed the structures of the arginine-insensitive NAGK of Saccharomyces cerevisiae (yNAGK). This enzyme, which is produced in yeast mitochondria by proteolytic processing of the polyprotein precursor (Uniprot database code Q01217) encoded by the ARG5,6 gene, has the unexplained peculiarity, which is shared by the NAGKs of other ascomycetes, of having, in addition to the AAK domain, a C-terminal domain of ∼150 residues of unknown structure and function (domain of unknown function) of the Pfam (http://pfam.sanger.ac.uk) and InterPro (http://www.ebi.ac.uk/interpro) databases. Another very interesting characteristic of yNAGK is that it forms with yeast NAGS (yNAGS) a complex with the features of a true metabolon Xanthomonas campestrisWe report here structural and functional studies on the arginine-sensitive NAGK from function  [10]–. In  subunit  differ f subunit . As alreP. aeruginosa NAGK) consists of a protruding, slightly kinked helix that interlaces with the same helix of another subunit from a different dimer  and a rotation of 25° about its central axis . These carginine , this diwww.pdb.org) accession codes 3ZZI and 4AB7). Although the resolution attained was 3.25 Å at the most, the high quality of the averaged electron density map (illustrated in the 351GYK) (Neisseria gonorrhoeae NAGS (NgNAGS) is associated with a rotation of approximately 109° of the C-terminal domain of this enzyme around the linker connecting it to the AAK domain Two closely related, yet non-isomorphous crystals of full-length mature yNAGK, each containing two enzyme tetramers in the asymmetric unit, have allowed determination of the structure of complete yNAGK using X-ray diffraction ( 351GYK) . It is im values for the substrates. This lack of effect on the apparent affinity for both substrates reflects the lack of physical interactions of the DUF619 domains with the substrate sites, which remain equally exposed in the structures of the truncated  at the entrance of helix D′, is replaced in yNAGK by 431WLNNVT436 (the motif-characteristic residues are underlined)  open αβα sandwich nucleated by a 7-stranded antiparallel β-sheet, which has the characteristic architecture of the histone acetyltransferase fold (GNAT fold)  [16]. Therlined) . T436 reerlined) . In cont2 of extra buried surface in the whole enzyme tetramer, and should, thus, substantially contribute to tetramer stability. The plasticity of the “stands” at the dimeric junctions, together with the rigidity of the pairing of the DUF619 domains, should restrain the mobility required by the opening/closing of the active centers in the AAK domains.The pairing of the DUF619 domains on each side of the yNAGK tetramer involves hydrophobic interactions between the E′ and F′ helices as well as the continuity of the central β sheets by way of antiparallel bonding of strands 6′ from both domains . These iX. campestris, with apparently the same domain organization as yNAGK and a high NAGS activity The present work raises questions as to why yNAGK has an inactive GNAT domain and a distinctive tetrameric architecture. Identification of a bifunctional NAGS/NAGK enzyme in The present results may also be relevant for human NAGS, whose inborn errors cause clinical hyperammonemia 6 His-tag, the DNA sequence encoding enzyme residues 58–513 . yNAGK was truncated after residue 356 by replacing in the yNAGK-encoding plasmid codon 357 by the TGA stop codon, using the site-directed mutagenesis QuickChange kit (Stratagene), and primers 3 and 4 (Saccharomyces cerevisiae (strain FY250) genomic DNA  and inserted at the NheI/BamHI sites of pET28a. This construct fuses the His-tag including a thrombin cleavage site, MGSSH6SSGLVPRGSHMAS, to residue 353 of yNAGK, which is the first of the DUF619 domain. All the constructs were verified by automated DNA sequencing.To produce yNAGK which lacks the mitochondrial targeting sequence and is preceded by a MGH–513 see  was PCR- 3 and 4 . The DUF 3 and 4  from SacE. coli BL21 (DE3) grown to OD600 = 0.5–0.6  in 0.5–1 L LB medium, supplemented with 0.1 mg/ml ampicillin and then induced for 20 hours at 30°C with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). The Se-Met-substituted AAK domain was produced in the same way by replacing LB with SelenoMet Medium , adding to this medium 15 min before IPTG induction an amino acid mixture containing 60 mg/L L-Se-methionine, 100 mg/L of each, L-lysine-HCl, L-phenylalanine and L-threonine, and 50 mg/L of each, L-isoleucine, L-valine and L-leucine The complete enzyme and its AAK domain were expressed in 2, 1 mM dithiothreitol and either 0.25 M (complete yNAGK) or 0.5 M (AAK or DUF619 domains) NaCl, and the supernatant of the centrifuged sonicate was applied to a 1-ml HisTrap column fitted in an ÄKTA FPLC . This column had been equilibrated with the sonication buffer enriched with 20 mM imidazole, and was washed successively with 5 ml of the same solution and then with 15 ml of 70 mM imidazole-containing solution. The pure proteins, eluted by increasing the imidazole concentration to 0.5 M, were concentrated to ∼10 mg/ml and freed from imidazole by centrifugal ultrafiltration.Subsequent steps were performed at 4°C. After centrifugation, cell pellets were sonicated in 1/25 of the original volume of a solution containing 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM CaCl2 and 0.25 M NaCl, collecting the tag-free protein by passing through a His-trap Ni-affinity column according to the kit instructions. Following confirmation of tag removal by SDS-PAGE  in 50 mM Tris-HCl pH 8.0, 10 mM MgClFor crystallization purposes, the surface-exposed lysine residues of the complete enzyme were reductively dimethylated 2 were fixed at 20 mM and 30 mM, respectively. When ATP was varied, the NAG concentration was fixed at 80 mM and that of MgCl2 was kept in 10 mM excess over that of ATP.NAGK activity was determined colorimetrically in the presence of 0.4 M hydroxylamine 2. The reaction was initiated with the enzyme and was stopped after 30 min by a 40-fold dilution with 0.2 mM Ellman's reagent in 0.1 M Na phosphate, pH 7.0, determining absorbance at 412 nm.NAGS activity was determined at 37°C as glutamate-triggered CoA release from AcCoA by monitoring colorimetrically SH group production with Ellman's reagent 2, 6 mM of the inert ATP analog adenosine 5′- triphosphate (AMPPNP) or 2 mM L-arginine-hydrochloride, added individually or as mixtures. The DUF619 domain, either with or without His-tag, yielded no crystals. The best crystals of the truncated enzyme (native or Se-Met substituted) had approximately 0.4 mm largest dimension and grew in 24-well plates at 4°C in 2-µ1 hanging drops composed of 1/1 24 mM NAG-containing protein solution/crystallization solution . The crystals, harvested in crystallization solution supplemented with 25% glycerol, and, in the case of the native NAG-containing crystals, with 2 mM HgCl2,  were frozen in liquid nitrogen and used for diffraction data collection at 100 K (Oxford Cryosystems) at the ESRF synchrotron (Grenoble). One crystal was soaked with arginine by adding 0.5 µl of 0.2 M L-arginine-hydrochloride to the drop 24 hours before harvesting. Another crystal was freed from NAG by successive three 2-hour passages through 5 µl drops of NAG-free crystallization solution prior to harvesting in the cryobuffer and freezing.1 belonged to one type and diffracted X-rays at 3.1 Å-resolution. It grew at 4°C in sitting drops composed of 0.4 µl of reductively methylated protein solution without ligands and 0.4 µl crystallization solution containing 0.2 M NH4I and 20% PEG3350 . Crystals NAGK2 and NAGK3  were of the same type, and grew in sitting drops at 4°C in the presence of 40 mM NAG using a crystallization solution containing 0.2 M ammonium citrate pH 7.0 and 12% PEG3350 . Crystallization solutions with PEG3350 concentration raised to 40% were used as a cryobuffer for liquid nitrogen freezing.Three crystals of the complete enzyme were used for data collection. Crystal NAGKP212121) contained four subunits in the asymmetric unit and 49% solvent content (1 of the complete enzyme had a monoclinic cell (space group C2) with two subunits in the asymmetric unit, whereas crystals 2 and 3 had triclinic cells (space group P1), with eight subunits in the asymmetric unit.The data for the crystals of the truncated enzyme were processed with MOSFLM  content . Crystalfree. TLS (translation/libration/screw) 1 crystal by molecular replacement with PHASER using one subunit of the truncated enzyme as search model to yield a dimer in the asymmetric unit. As refinement progressed, electron density became clearer for the DUF619 domain of the two subunits, and a partial model (about 75% of the domain main-chain) was built. This incomplete NAGK1 model was then used to obtain an initial solution for the NAGK2 crystal by molecular replacement with MOLREP, consisting of 4 dimers forming two tetramers in the asymmetric unit. Further phase improvement was achieved by density averaging between the NAGK crystals 1 and 2, using DMMULTI of the CCP4 suite 2 structure , the quality of the maps allowed the recognition of most of the side chains in the DUF619 domain. The third dataset, collected to 3.25 Å resolution , yielded eight subunits in the asymmetric unit forming two tetramers. The model, including two NAG molecules, was built and refined to 3.25 Å resolution, as for the NAGK2 crystal, except for the N-terminal helix of four subunits (two per tetramer at the interdimeric junction), which presented poor density.For the complete enzyme, initial phases were obtained for the 3.1 Å dataset collected from the NAGKtructure . Howeversolution  solved b2 and NAGK3 crystals, yielded good Rfactor/Rfree values and showed good stereochemistry (monitored with PROCHECK) http://www.pymol.org).The final models for the crystals of the truncated enzyme without NAG, with NAG, and with arginine, and of the complete NAGK0.5 is the temperature giving a fluorescence increase corresponding to 50% of the maximum increase.Thermal stability was assessed with the thermofluor approach Analytical size exclusion chromatography through Superdex 200  was carried out at 24°C, as reported elsewhere www.pdb.org).The atomic coordinates and structure factors of yNAGK lacking the DUF619 domain, complexed with NAG (PDB ID: 3ZZF), NAG and arginine (PDB ID: 3ZZH), or ligand-free (PDB ID: 3ZZG), as well as those of the complete enzyme (PDB ID: 3ZZI and 4AB7), have been deposited in the Protein Data Bank  and EcNAGK (B). In both dimers, the subunit on the left (lighter) is fixed in the same orientation, to highlight the different relative orientation of the other subunit in both NAGKs, in relation to the fixed subunit. The N-terminal “stand” of yNAGK has been omitted for clarity.(TIF)Click here for additional data file.Figure S2Stereo view of the interactions of one “stand” with its subunit body (both brown) and with the other “stand” (pink) across the interdimeric junction.(TIF)Click here for additional data file.Figure S3Experimental information for the DUF619 domain. (A) Stereo views of the 2Fo-Fc (blue grid) and Fo-Fc (red grid) electron density maps of the complete DUF619 domain (shown as Cα), contoured at 1.0 and 2.5 σ, respectively. (B) Detailed view of a region of the domain, with the model in sticks representation , to illustrate that the map quality allows identifying most amino acid side chains (labelled for some residues).(TIF)Click here for additional data file.Table S1Oligonucleotides used in cloning and site-directed mutagenesis.(DOC)Click here for additional data file.


We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times. Protein domains are important building blocks of proteins. They often act as the common currency of protein function that nature rearranges to create novel activities Fusobacterium nucleatum sub spp vincentiiDuring the construction of the Pfam database of protein domains etal. who published a preliminary version of Jackhmmer The YARHG domain was initially identified using the Jackhmmer program, which is part of the HMMER3 package written by Sean Eddy Predictions of the secondary structure of the domains were carried out using the Jpred3 software To aid in discovery of the function we attempted to identify other domains that are associated on the same sequence with the YARHG domain. To identify associated domains we searched all the proteins found in the family against the Pfam database.http://www.atgc-montpellier.fr/phyml/, to generate a bootstrapped (100) phylip tree in Newick-format. For the same sequences a corresponding phylip-interleaved file of all the domain architectures was produced manually. These two files were run together through the iTOL web-browser at: http://itol.embl.de/upload.cgi, to produce a phylogenetic tree with associated domain-architectures.A phylogenetic tree was generated from a representative sequence-alignment using Phymol 3.0 with default parameters: We initiated the Jackhmmer search with the uncharacterised protein-product of gene FNV1478 (UniProt Knowledgebase accession: Q7P768.1), using default parameters, against the UniProtKB database The conserved region of the YARHG domain is 70 amino acids in length and shows a number of conserved residues that are likely to be important for its structure and function. In addition to the YARHG motif there is a highly conserved C-terminal glutamate residue as well as two pairs of conserved aromatic amino acid residues. Although the conserved charged arginine (from the YARHG motif) and the glutamate might be suggestive of an enzymatic domain, it is unusual for such short domains to carry out any enzymatic activity. The secondary-structure predictions identify a set of four alpha-helices see , suggestThe YARHG domain is an extracellular domain. In almost every case it is found in a protein that possesses a signal-peptide, targeting the protein for secretion, and/or contains one or more trans-membrane helices, with the YARHG domain predicted to be extracellular. One of the few exceptions to this is in the original query protein FNV1478. Alignment of this protein to its close relatives shows that it is truncated at the N-terminus relative to its homologues that possess a predicted signal peptide. We thus suggest that the initiating methionine of the FNV1478 protein-product has been incorrectly predicted.Acinetobacter sp. strain ADP1 (UniProtKB accession: Q6FA13_ACIAD), finds only other YASKG- and YARHG-carrying sequences. Although these proteins are quite distinctive in sequence from the classical YARGH domain members their predicted secondary-structure is comparable  of identified proteins. One feature of the YARHG domain is that it is very frequently found at the C-terminus of proteins, and most of such proteins have a signal-peptide at their N-terminus.E. coli uses an extracellular domain to sense changes in the external pH, signalling this through the membrane, is the case of CadC. CadC, a membrane-integrated transcriptional activator, is released only at low pH when it then binds directly to the DNA in the cadBA operon and alters gene expression The YARHG domain is frequently found (in 73 instances) associated with a pair of N-terminal zinc-ribbon domains . Zinc-ribbon domains are often involved in nucleic acid-binding There are six sequences where the YARHG domain is associated with a protein serine/threonine kinase (Pkinase) domain (Pfam accession: PF00069). Four of these proteins come from Gram-negative Cyanobacteria, see example A0ZHA2. These four sequences are all very similar. They are approximately 500 residues long with no signal-peptide; however, they all have a trans-membrane helix between the N-terminal Pkinase and the C-terminal YARHG domain, suggesting that the YARHG domain is extracellular. None of them has any other associated domains.The remaining two sequences carrying a Pkinase domain are from Gram-positive Firmicutes, see D4L881 and B0MGE2 in In all of these six cases the architectural arrangement hints at some form of cross-membrane signalling, whereby the YARHG might be detecting some external ligand whose binding triggers the Pkinase to phosphorylate downstream (intracellular) protein substrates. The PASTA domain binds to beta-lactam antibiotics and is suggested to bind to unlinked peptidoglycan cross-links The YARHG domain is, additionally, found in a single protein (E1L326) associated with a SPOR domain (Pfam accession: PF05036). This domain has also been shown to bind to peptidoglycan.Bradyrhizobium sp contains a Caspase-like cysteine-aspartic protease (Pfam accession: PF00656) with a pair of tandem repeated YARHG domains at the C-terminus. This protein has a signal-peptide so is secreted. This is the only sequence from this nitrogen-fixing Bradyrhizobium genus to carry a YARHG domain Three sequences from different species, from different bacterial clades , carry an associated peptidase domain, although the peptidase-type is different in each case. The UniProtKB sequence accession A5ECX2 from E. coli Htpx protein has three trans-membrane regions whereas our M48 example, C7N9T6, has five, suggesting that it might be behaving slightly differently. Gram-negative bacterial proteases may be involved in regulated intra-membrane proteolysis (RIP) Metallopeptidases of the MEROPS M48 family Marvinbryantia formatexigens, C6LL79, example in Peptidase family S8, also known as the subtilisin family (Pfam accession: PF00082), is the second largest family of serine peptidases (MEROPS). Members of family S8 have a catalytic triad in the order Asp, His and Ser in the sequence, and have a characteristic GTSMA sequence-motif also found in the Firmicute sequence from Two Clostridial sequences each carry a long Periplasmic-binding protein superfamily domain Marvinbryantia formatexigens carry a pair of SH3 domains (Pfam accession: PF08239). This bacterium is a Clostridial gut acetogen with the ability to consume oligosaccharides and boost the yield of succinate Two sequences from Marvinbryantia formatexigens, the Clostridial gut acetogen with the ability to consume oligosaccharides One sequence from Fusobacterium spp carry four MORN  repeats in tandem at their very N-termini, the YARHG domain in each one then lying extra-cellularly at the very C-terminus, separated from the MORNs by a single trans-membrane helix. These repeating sequence-motifs are found in junctophilins Two sequences from The species carrying the YARHG domain are distributed fairly evenly between Gram-positive and Gram-negative bacteria, with 50% being Firmicutes, the remainder being a mixture of Gram-negative Proteobacteria –20%– Fusobacteria –13% – Bacteroidetes –10%– and a handful of others including 3.5% Spirochaetes. The proteins that possess the exact YARHG sequence-motif are almost all Gram-positive, whereas the cysteine-containing YASKG subfamily member-species are all Gram-negative. To examine the phylogenetic distribution of the YARHG domain between the Gram-positive and Gram-negative bacterial species that carry it, we have produced a combined phylogeny and domain-architecture diagram as shown in Phylogenetically, the Gram-positive and Gram-negative sequences are not clearly separated in the tree, but one should bear in mind that the analysis was run on just the short section of each sequence contained only in the YARHG domain. However, there are still large clades that contain only Gram-positive or Gram-negative sequences. By making multiple sequence-alignments of each type we noted that the Gram-positive YARGH domains conserve the YARGH sequence-motif much more strongly than the Gram-negative examples do.The phylogeny of the YARGH domain proteins suggests that although many different domains are found associated on these sequences it is likely that most if not all domain-fusion events happened only on a single occasion in evolution. All sequences carrying the zinc-ribbons are on Gram-positive sequences, and their current distribution might suggest that all their more closely neighbouring sequences once carried such an N-terminal domain but that this has subsequently become lost over time along some lineages. The Peptidase domain is the only independent domain found on both Gram-positive and Gram-negative sequences, but in each of the three cases the peptidase is of an evolutionarily different class, a metallo-peptidase for C7N9T6, a cysteine-peptidase in A5ECX2 and a serine-peptidase for C6LL79. In general, any individual corresponding domain is associated with just one or at most two distinct clades, e.g., the group of Gram-negatives at the top of A question arises as to the mechanism of domain-gain in YARGH proteins. The two examples, of YARHG with DUF4453 among some Gram-negatives and of YARHG with DUF4454 among a small group of Gram-positives see , are intIn this study we have identified a new protein domain that has been associated with a wide variety of other protein domains during evolution. Although the functional nature of this domain is uncertain we hypothesise that it binds to an as yet unknown ligand. We think that the most likely candidate for such a ligand is the peptidoglycan component of the bacterial cell wall. The domain is found in Gram-positive bacteria that have a thick peptidoglycan layer. In these organisms the domains strongly conserve the YARHG motif. In Gram-negative bacteria, which have a thinner peptidoglycan layer, the YARHG motif is much less well conserved suggesting that the domain might have either a more diverse range of ligands or that it no longer binds a ligand. Future functional and structural studies will be needed to elucidate in full the role that this domain plays.


The availability of genome sequences for some of the most devastating eukaryotic plant pathogens has led a revolution in our understanding of how these parasites cause disease, and how their hosts respond to invasion Hyaloperonospora arabidopsidis, Phytophthora infestans, and Phytophthora sojaePhytophthora RXLR effectors contain conserved sequence motifs  in their C-terminal domains that often form tandem repeats One expanded family of effector proteins is defined by the sequence RXLR , which in some cases is followed by an acidic-rich dEER motif (Asp-Glu-Glu-Arg) . The RXLPhytophthora capsici), PexRD2 (from P. infestans), and ATR1 (from H. arabidopsidis) Our laboratories have employed structural biology to investigate the molecular basis of RXLR effector function. A total of four structures have recently been published, those of AVR3a4 and AVR3a11 . Using Hidden Markov Model (HMM)-based sequence searches, these motifs had also been detected in H. arabidopsidis RXLR effector proteins, with 35 out of 134 (26%) containing this fold (HMM score>0), including ATR1 with a low confidence score The studies of Boutemy et al. and Chou et al. independently described the structural homology of AVR3a11 and a domain of ATR1, respectively, to the cyanobacterial four-helix bundle protein KaiA The availability of these four oomycete RXLR effector domain structures, from three different pathogens, allows us to present a detailed analysis of the WY-domain fold. Structural overlays of the conserved WY-domains from each of the effectors are shown in Saprolegnia parasiticaAlbugo labachiiPythium ultimumPhytophthora and Hyaloperonospora proteomes searched previously P. infestans>P. sojae>P. ramorum>H. arabidopsidis) also encode a significantly higher percentage of WY-domains in their secretomes  and Hyaloperonospora (obligate biotrophs), but not P. ultimum, it appears that the WY-domain emerged with biotrophy in this lineage along with the evolution of haustoria and RXLR effectors We believe that the conservation of this “flexible” hydrophobic core fold indicates that a large family of plant pathogenic oomycete effectors may have been derived from a common ancestor. Intriguingly, this effector family has rapidly diverged to gain new and/or adapt existing virulence functions and/or evade detection by plant immune systems. New or modified effector functions may be derived from surface point mutations or indels in the connecting regions between helices (including domain duplication). To test the argument for a common ancestor, we extended previous analyses and searched the proteomes of various organisms using the HMM for the WY-domain, as described in Some of the most notorious and agriculturally important pathogenic oomycetes contain RXLR:WY-domain effectors, suggesting that this structure has been critical for the success of these pathogens. This raises questions such as, why has this fold been preserved and what can it tell us about the function of these proteins? How can we use this knowledge to design novel disease management strategies? Future studies will help define the roles of the WY-domain fold in the virulence mechanisms of these pathogens, in particular how it engages with plant cell targets, and will help to unravel the extent of structural diversity in RXLR effectors. Our initial studies have laid the foundation for new, exciting discoveries addressing the function of oomycete effectors.


DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain.DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a CysHere, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501–544 determined by NMR spectroscopy. The structure adopts a ββα fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners  seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins. Mutations in these proteins are linked to cancer and other pathologies. DEAF-1 is an important transcriptional regulator that is required for embryonic development MYeloid translocation protein 8, Nervy and DEAF-1) domain Epstein-Barr virus-induced Nuclear Antigen2) protein. These interactions depend on the presence of a PXLXP amino acid motif (X: any amino acid). Interestingly, whereas MYND domains in the Bra-1 and Bra-2 proteins were found to bind E1A and EBNA2, no interaction of the RACK7 or ETO MYND domains with these viral proteins was observed The DEAF-1 protein contains two conserved domains, the SAND  domain, originally named KDWK after the conserved amino acid core, and a cysteine rich region called the MYND  of 5.30±0.54 mM and 3.08±0.12 mM for SMRT and NCoR peptides, respectively. NMR 15N relaxation rates measured in the absence and presence of a large excess of peptide show no significant changes in dynamic properties (data not shown). This confirms that ligand binding does not affect the oligomerization of the domain as expected (see above).Although the DEAF-1 MYND domain adopts a ββα fold common to many DNA binding zinc finger proteins, it failed to interact specifically with DNA E. coli for structural and biochemical studies. However, based on the sequence conservation (∼40% identity) between the two domains, we calculated a homology model for the BS69 MYND domain based on the DEAF-1 structure using MODELLER in vitro translated GST-tagged BS69 protein, encompassing the MYND domain. As expected, mutation in one of the zinc coordination sites, completely abolishes the binding to any partners In order to investigate whether the structural characteristics and ligand interactions of MYND domains are conserved we studied the BS69 MYND domain. This domain was previously shown to mediate binding to the NCoR corepressor The solution structure of the DEAF-1 MYND domain reported here reveals a tandem C4-C2HC zinc-binding motif with a cross-brace topology. This structure corrects our previous report that was based on a C-terminally extended, longer construct of DEAF-1 MYND domain (residues 498–565), for which only few long range NOEs could be obtained ,b [25]. Our NMR titrations using peptides derived from the silencing mediator of retinoid and thyroid receptor (SMRT) and from the nuclear receptor corepressor (NCoR), show that the binding site of DEAF-1 MYND for these corepressors resembles that of the ETO MYND domain. The interaction of these ligands to DEAF-1 MYND is about an order of magnitude weaker than the one previously reported for the ETO MYND domain Recently, Foreman and coworkers determined the crystal structure of a multi-domain construct of SMYD3 and found that an intact MYND domain fold is required for interaction with the NCoR corepressor Conservation of critical residues involved in the interaction may thus explain why an interaction with corepressors is widely observed among the MYND domain family. While binding to corepressors such as SMRT and NCoR seems to be a common feature of many MYND domains, including those of ETO, BS69, and BOP Although BS69 has been shown to bind to NCoR Collectively, these data suggest that variations in amino acid sequence drive distinct binding specificities of MYND domains with their cellular and viral partners, even though binding to corepressors such as NCoR and SMRT seems to be a feature that is generally shared by many MYND domains. The relatively weak interaction of DEAF-1 MYND with the corepressor-derived peptides, may be further enhanced by additional protein-protein contacts involving different regions of the full-length DEAF-1 and corepressor proteins. Thus future functional studies of the role of DEAF-1 should consider the possibility that additional domains may further enhance the interaction. The DEAF-1 and ETO MYND domains appear to use the same binding mode when interacting with corepressors, and both fail to bind to MGA, E1A and EBNA2 proteins. In contrast, MYND domains that are more distantly related, such as BS69, may favor electrostatic interactions through their specific charged residues. Future structure/function studies will further highlight the importance of MYND-mediated protein-protein interactions in transcriptional regulation with viral and cellular proteins.AAACCATGGAGCAGTCCTGCGTTAAC containing NcoI site and the reverse primer: AAAGGTAGGTCATGCTGACTGGCCGCATATG containing KpnI. site were designed to clone MYND domain into the expression vector pETMThx, which was modified from pET24d to include an N-terminal poly-histidine tag, thioredoxin as a fused protein and a TEV cleavage site.The DNA fragments comprising the human DEAF-1 MYND domain (residues 501–544 and 496–565 for the short and long constructs respectively) plus an N-terminal insertion of three amino acids GAM, were PCR-amplified from Human cDNA. The forward primer: E.coli strain BL21(DE3). To prepare 15N, 13C and/or 15N isotope-labeled proteins expression was carried out in M9 medium containing [U-13C] glucose and/or [U-15N] NH4Cl as sole carbon and nitrogen sources. Overnight cultures were grown in LB medium at 37°C, diluted 50 fold into the M9 medium, continuously grown at 37°C to an OD600 nm of 0.6. The culture was induced with 1 mM IPTG. During the induction 0.3 mM ZnCl2 was added and the culture was switched to 20°C for 10–16 hours. All media contained 50 µg/ml kanamycin.The DEAF-1 MYND constructs were transformed into 2+-NTA , equilibrated with the lysis buffer. The bound proteins were eluted with 400 mM imidazole in the lysis buffer. The N-terminal His-tag and the fusion thioredoxin were removed by adding 1∶50 (molar ratio) TEV protease, supplemented with 2 mM DTT to the pooled protein solution. After removing the residual DTT, using a desalting column, PD10 , the TEV protease and cleaved His-tag were removed by an additional Ni-chelating affinity chromatography.Bacteria cells were harvested by centrifugation at 8,000 g for 30 min and re-suspended in 3 ml/g (wet weight) lysis buffer , supplemented with protease inhibition cocktail complete  and DNase I (10 µg/ml). Cells were lysed by pulsed sonification . Cell debris was removed by centrifugation at 15,000 g for one hour. All proteins were purified by two steps of chromatography. The supernatants were applied to NiThe flow through from the second Ni-chelating affinity chromatography was collected, concentrated and loaded on to a gel filtration HiLoad Superdex 75 16/60 column  previously equilibrated with 50 mM phosphate buffer, pH 6.5, containing 100 mM NaCl, 2 mM DTT. The fractions containing MYND proteins were pooled and concentrated to 0.3–1 mM for NMR measurements.15N/13C labeled sample in 90% H2O/10% D20 15N- and 13C-edited NOESY spectra were recorded on an Avance 900 spectrometer equipped with a TXI cryo-probehead with a mixing time of 80 ms (100 ms for the aromatic 13C-edited NOESY spectrum). Data were analyzed using Sparky The chemical shifts of the DEAF-1 MYND domain were assigned using standard heteronuclear experiments acquired at 295 K on a 1 mM uniformly 2H quadrupolar splitting of ca 20 Hz. Hα-Cα, HN-C′, N-C′ and HN-N residual dipolar couplings (RDCs) were recorded on a 600 MHz spectrometer equipped with a TCI cryoprobehead using experiments described elsewhere A 50 mg/ml phage solution was used to weakly align the sample in an anisotropic phase, resulting in a 15N longitudinal (R1), transverse (R2) relaxation rates, and the {1H}-15N heteronuclear Overhauser (NOE) effect were carried out on the in-house 750 MHz spectrometer equipped with a TXI probehead using standard methods 1 and R2 relaxation rates, ten different time-points were recorded in an interleaved manner. Peak intensities were fitted to a two-parameter exponential decay using NMRViewJ NMR relaxation measurements including 15N-labeled MYND domain was titrated with peptides SNPPPLISSA (SMRT) and RPPPPLIPSS (NCoR) up to a 9-fold and 5-fold molar excess, respectively. The changes in 1H and 15N chemical shifts are referred to as chemical shift perturbations (CSP) and were calculated as:Dissociation constants for the interaction of DEAF-1 MYND with proline-rich peptides derived from SMRT and NCoR corepressors were determined from fitting chemical shift changes monitored in NMR titration experiments to a binding isotherm. A 1 mM solution of max is the chemical shift perturbation at saturation, [TP] is the total protein concentration, and DK, the dissociation constant.The CSPs were fitted to a binding isotherm using the equation:2+-coordination, torsion angle restraints derived from chemical shifts using TALOS+ γ 2.3 Å and Zn-Nε2 2.0 Å) and angle  restraints. This procedure ensures a proper tetrahedral zinc coordination, which is not fulfilled in most of the NMR-derived zinc-binding folds found in the Protein Data Bank. In this final step a total of 100 structures were calculated from which the twenty lowest energy structures were used for quality and structure validation using the iCING web interface (http://nmr.cmbi.ru.nl/icing/) and PROCHECK www.pymol.org).First structure calculations were performed with CYANA Mutations were performed using the QuickChange mutagenesis kit (Stratagene). Wild type and mutants BS69 GST- fusion proteins encompass the 150 last C-terminal residues of the protein (BS69Δ431) Figure S1Dependence of the MYND domain fold on zinc coordination. a. Overlay of 1H 1D spectra obtained from the wild type protein in the absence (red) and presence (cyan) of EDTA. b. Overlay of 1H-15N HSQCs of the wild type protein (red) and H538S mutant (cyan).(TIF)Click here for additional data file.Figure S2Oligomerization of DEAF-1 MYND domain. a. Left: Continuous sedimentation distribution obtained for 86 µM DEAF-1 MYND domain. Analysis revealed a 9.95 kDa species suggesting that MYND monomers (5.2 kDa) associate into dimers. The monomer and dimer appear to be in a in rapid monomer-dimer equilibrium (data not shown). Right: Concentration-dependent sedimentation velocity analysis of DEAF-1 MYND domain. Dimerization occurs at sub-micromolar concentrations. The data were fitted to a self-association model yielding an estimated KD of ∼0.5 µM (±0.1 µM). b. Overlay of 1H-15N HSQC spectra recorded at 1 mM (black) and 50 µM (cyan) protein concentration. Cross-peaks that experience line-broadening upon dilution are annotated with the corresponding residue numbers. c. Cartoon representation of the DEAF-1 MYND domain. Residues affected by the dilution of the sample are clustered around the first zinc binding site and are shown as cyan sticks, whereas those involved in binding to co-repressor peptides are shown as magenta sticks. Residue H538 is also shown in black.(TIF)Click here for additional data file.Figure S3Salt and pH dependance of oligomerization of DEAF-1 MYND domain. a. Overlay of 1H-15N HSQC spectra (left) and long-range 1H-15N HSQC spectra (right) recorded at a NaCl concentration of 100 mM (black) and 400 mM (green) b. Overlay of 1H-15N HSQC spectra (left) and long-range 1H-15N HSQC spectra (right) recorded at a pH of 6.5 (black) and 6 (red). At pH 6, H538 tends to a conformation where both nitrogens of the side chain, are protonated, thus excluding any possible ion binding. c. 15N R1 and R2 relaxation rates of DEAF-1 MYND domain for three different samples, i.e., [NaCl] = 100 mM and pH = 6.5 (black), [NaCl] = 400 mM and pH = 6.5 (green), [NaCl] = 100 mM and pH = 6 (red). No significant change in relaxation rates are observed changing the buffer conditions. The oligomerization state revealed by the high R2/R1 ratio is thus likely due to non specific interactions.(TIF)Click here for additional data file.Figure S4Correction of the previously reported structure of DEAF-1 MYND domain. a. Cartoon representation of the corrected DEAF1-MYND structure with long-range NOEs from the new NMR data. Side chains of residues showing long-range NOEs are shown in as magenta sticks. Newly observed long-range NOEs, which define the cross-brace zinc binding topology, are indicated by green lines. Long range NOEs that were also observed previously are shown as dotted black lines. b. Comparison of original and new NOESY experiments showing important NOEs originating from aromatic residues. Left: The NOE contacts of the His 536 Hε1 proton in a homonuclear 2D NOESY  and in the 3D aromatic 13C edited NOESY spectrum . Right: The same for NOEs involving the Tyr 523 Hε1 proton. NOEs observed only with the new experiments are green boxed.(TIF)Click here for additional data file.Figure S5Binding of E1A to wild-type or mutant BS69 proteins. The effect of individual mutation of residues RRKR 559–562 of Bs69 for E1A binding was tested. 2× 106 QT6 fibroblasts were transiently transfected with 12SE1A and wt or mutant FLAG-tagged BS69 411–561 peptides as notified on top. 24 h post-transfection, protein lysates were immunoprecipitated with a 10 µg anti-FLAG antibody in a 100 mM NaCl, 20 mM Tris pH8, 0.5% NP40 buffer and Protein A sepharose beads. After extensive washes in the binding buffer, proteins were eluded from beads and separated by SDS-PAGE. Immuno-precipitated E1A protein was revealed by western blotting. Individual mutations of these residues indicate that among these four residues only Arg560 is essential for binding of BS69 to E1A.(TIF)Click here for additional data file.