Anchistioides antiguensis, all species examined possessed cuticular structures at the distal end of the pars molaris (molar process). Five types of cuticular structures are recognised herein, each with a unique form, but variable in number, placement and arrangement. Each type is presumed to have a different function which is likewise related to diet.The mandibles of caridean shrimps have been widely studied in the taxonomy and functional biology of the group. Within the Palaemonoidea the mandibles reach a high level of structural diversity reflecting the diverse lifestyles within the superfamily. However, the majority of studies have been restricted to light microscopy, with the ultrastructure at finer levels poorly known. This study investigates the mandible of nine species belonging to six of the recognised families of the Palaemonoidea using SEM and analyses the results in a phylogenetic and dietary framework. The results of the study indicate that little phylogenetic information is conveyed by the structure of the mandible, but that its form is influenced by primary food sources of each species. With the exception of  Palaemon elegansPalaemon, using light microscopy to examine its structure and function (Leander) and interspecific variation  and pars molaris (molar process) and may be provided with a palp or not. Both the pars incisivus and the pars molaris are variable in form ranging from truncated to elongate, straight to markedly curved, narrow to flared, widely separated to barely separated and many gradations in between , with details included in terature . DespiteThe methods used for preparation of tissue follow those established by pars molaris refers to their position in situ , with the distal segment being more slender and slightly longer than the basal and penultimate segments. Distally-serrulate setae are present , oval  in cross-section. Most are roughly parallel sided but those of H. picta and G. elegans are strongly curved, that of D. bislineata has convex lateral margins and in A. antiguensis the pars molaris is strongly flared distally. Teeth are present distally on most mandibles  ted plate –6F. The menocera  and 4F, ophyllum  and morepars molaris of most mandibles examined here were found to be covered, to a greater or lesser degree, by numerous filamentous structures, which are flexible to semi-rigid and frequently developed into rows  can be recognised, which appear to relate to feeding mode or diet, although five of these types apply to single species only and the link with specialised food resources would require greater taxon coverage to include other species that share similar diets.Type A mandible: Well developed pars incisivus and pars molaris; pars molaris distally cuspidate; with Type I CS; encountered in Palaemon macrodactylus, Macrobrachium nipponense, Euryrhynchus wrzesniowskii and Pontonia pinnophylax  or material collecting in the mantle cavity. Similarly, Pontonia mexicana  to hold and manipulate food  and G. americanum  that it would be reasonable to assume a similar diet. However, Hymenocera feed exclusively on starfish, particularly Nardoa and Linkia spp. piercing the epidermis with their first pereiopods before extracting internal tissues.Gnathophyllum. It seems likely, therefore, that the Type E mandible is a further development of the Type D mandible in response to a dietary switch in Hymenocera (or its ancestors) from merely removing mucus from the echinoderms to actually predating on them. The paired teeth of the right pars molaris apparently interlink with those of the left and may take on the slicing role normally attributed to the pars incisivus.The sparse arrangement of cuticular structures would also not be as effective at moving mucus or particles as those in the Type D mandible of Type F mandible: Well developed pars incisivus and pars molaris; pars molaris distally flattened and ridged; with Type V CS; only encountered in Desmocaris bislineata.pars molaris. They appear to be flexible and may be regarded as shorter versions of the cilia-like Type III CS. A particulate or detritivorous diet may therefore be expected. This is consistent with the information provided by pars incisivus is present for initial tearing, the Type F mandible does not have obvious grinding function and it is unclear how these carrion prey items would be broken down prior to ingestion. Another possible function for the elaborate arrangement of cuticular structures in this mandible type is that they may help to filter particular matter.Type V CS are the most highly developed of all the cuticular structures noted in this study. They in turn dictate the form of this mandible type as the finger-like projections together form the ridged surface of the pars molaris and pars incisivus, combined with a 3-segmented palp. Indeed, the recognition of several families, including some incorporated in this study, has partially been justified by the form of the mandible. The ridged nature of the pars molaris, which is presumed to be a primitive feature ; however, the other genera with this form of mandible are more difficult to explain from a phylogenetic point of view. Pontonia shares a greater affinity to Gnathophyllum, Hymenocera and Periclimenaeus , the mandibles of ides see . Mitsuhaolthuisi   the arrangement and form of the teeth would suggest that it does not grind at all. The form and arrangement of cuticular structures at the distal end of the pars molaris shows a particularly high degree of variation. The five types of cuticular structures recognised in this study are presumed to have different functions related to food sources, which is contrary to the findings of This study has demonstrated that the form of the mandible is much more complex than previously thought. The traditional view that the Some evidence of evolutionary relationships is conveyed through the broad structure of the mandible but the detailed structures witnessed in this study do not reflect the evolutionary relationships in the Palaemonoidea suggested by previous phylogenetic reconstructions . This pr


PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106–212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins.The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal  Chlamydia trachomatis is one of the leading bacterial pathogens, infecting over 100 million people worldwide annually [C. trachomatis is an obligate intracellular pathogen, with fifteen different serovars affecting various mucous membranes in the body. C. trachomatis invades epithelial tissues including those in the eyes and reproductive tract, where it affects the single outer layer of columnar cells. Currently, azithromycin and doxycycline are the antibiotics of choice to combat chlamydial infections. However, because infections are asymptomatic in nearly 70 percent of cases, patients frequently fail to seek treatment [C. trachomatis stands to pose a significant public health threat in the near future, similar to that currently posed the recent drug-resistant Neisseria gonorrhoeae.annually . C. tracreatment , 4. As areatment . Based oC. trachomatis has a biphasic developmental cycle, in which infectious elementary bodies (EB) invade host cells. From there, the resulting endosomal compartment evades lysosomal degradation pathways and forms an intracellular parasitophorous vacuole termed an inclusion. From within this inclusion, C. trachomatis is effectively cloaked and protected from host immunological defenses. Chlamydial EB differentiate into metabolically active reticulate bodies (RB) that undergo replication and initiate protein synthesis [C. trachomatis secretes effectors into the host cytoplasm that perform numerous functions to maintain the infected cell in a state where the immune defenses are dampened, cell viability is enhanced, and pro-apoptotic signaling is inhibited [ynthesis . During nhibited –10. The Chlamydia that cleaves host proteins including the cytoskeleton intermediate filament vimentin, the nuclear envelope lamin-associated protein (LAP1), and also disrupts cell division [Chlamydial effector proteins are translocated into the host cell cytoplasm and dynamically remodel the inclusion, interfere with host cell apoptosis, dampen immune responses, and inflammation, as well as abrogate or co-opt signal transduction pathways. Of the known chlamydial effectors, the serine protease chlamydial protease-like activity factor (CPAF) has been identified as a factor involved in the intracellular biology of division –15. HowePSD95 Dlg ZO-1 (PDZ), Src Homology 2 (SH2), Src Homology 3 (SH3), and WW domains [Assembly of many functional signal transduction complexes are facilitated by protein-protein interaction motifs such as  domains . In part domains –20. PDZ  domains , 22. PDZ domains –25. Addi domains .106-212 sequence is most similar to PDZ domains of human proteins involved in cell polarity and epithelial tight junction formation.While inspecting the CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PDZ domain. This PDZ domain is unique in that it bears almost no sequence similarity to canonical PDZ-forming sequences and displays minimal sequence and structural similarity to known chlamydial PDZ domains. We found that the CPAF106-212). The coordinates corresponding to residues CPAF106-212 were extracted from the mature CPAF structure and analyzed by the methods outlined below.All protein structures were retrieved from the RCSB Protein Data Bank repository . Upon in106-212 structure was compared to those of canonical PDZ domains known to bind to class I, II, and III ligands, as well as to those of unclassified PDZ domains (PDB ID): (Class I) PSD95-PDZ3306-402  [9-91  [112-193 and syntenin-PDZ2195-273  [1318-1412  [(Class II) CASK487-572  [69-153  [380-480  [362-454  [(Class III) nNOS14-100  [653-741  [1001-1096  [(Unclassified) PS2-D1P158-249  [287-379  [136-219  [6-92  [354-453  [The CPAF306–402) , NHERF2-195–273) , and erb77–1371) ; (Class  69–153) , HtrA138380–480) , and Htr01–1096) ;   and HtrA106-212 using the STructural Alignment of Multiple Proteins (STAMP) feature of the Visual Molecular Dynamics (VMD) software suite [106-212 to the representative canonical PDZ domains.Each set of PDZ domain coordinates was extracted from the full PDB coordinates and aligned to CPAFre suite , as wellre suite . Root me106-212 were submitted to the Dali Protein Structure Database server [The PDB coordinates for CPAFe server  in ordere server  to retriHomo sapiens, 13 (20%) from mouse and rat, 3 (5%) from Escherichia coli, and 2 (3%) from Drosophila melanogaster.The UniProtKB accession numbers for the resulting proteins were cross-referenced with the NCBI Conserved Domain Database (CDD)  to map a106-212 using the NCBI Protein BLAST (blastp) server [The 65 proteins mapped to 170 PDZ domain sequences, which were retrieved from the UniProtKB database and limited to the residue ranges provided by the CDD search. The PDZ domain sequences were aligned against CPAF) server , 48 to i) server . The res) server  for figuChlamydia. The chlamydial PDZ domains were cross-referenced against the UniProtKB database and the proposed function(s) for each protein was obtained from the database.We searched across all chlamydial strains and species for proteins containing annotated PDZ domains using the Pfam database . The res106-212). Canonical PDZ domains share a common fold consisting of five antiparallel beta strands and two alpha helices, in which the ligand-binding pocket is formed by the β2 strand and α2 helix, as depicted by the class I PDZ domain from PSD95 (PSD95-PDZ3)  . Variati2 domain . Additio2 domain .The residues lining the ligand-binding pocket of PDZ domains contribute significantly to peptide ligand binding and specificity. Thus, sequence similarity along the PDZ binding site hotspots is frequently a strong predictor of ligand specificity , 53. Can106-212) to representative structures of all three canonical PDZ domain classes, as well as to unclassified domains   to ident106-212, we aimed to address to what extent these domains retain sequence homology with the CPAF PDZ domain. The 170 PDZ sequences that were previously identified were retrieved from the UniProtKB database using residue ranges supplied by the NCBI CDD. These sequences were submitted for alignment against CPAF106-212 using the NCBI Protein BLAST (blastp) server [106-212. These sequences were submitted for multiple sequences alignment using the Clustal Omega webserver [Having identified a set of proteins containing PDZ domains with high structural homology to CPAF) server , 48. We ebserver , and theebserver  Fig 3)..106-212,106-212 within the PDZ domain recognition pocket. The conserved residues lie toward the core of the CPAF PDZ domain and binding pocket and may suggest that the residues are involved in establishing the PDZ fold to mimic host domains or in recognizing PDZ ligands  originate from C. trachomatis, making it the chlamydial species with the highest PDZ domain occurrence. Interestingly, all 40 of the PDZ-containing proteins identified in C. trachomatis are classified as hydrolases, in particular tail-specific and HtrA class peptidases.Having identified a cryptic PDZ domain in CPAF, we used the Pfam database to address whether PDZ domains are commonly annotated in chlamydial species and to which proteins they are coupled. We searched the Pfam database for known chlamydial PDZ domain types  and identified 86 unique PDZ-containing proteins from all species of 106-212 was shown to exhibit significant structural homology with unclassified PDZ domains, particularly that of human MPP7. The CPAF PDZ domain also shares structural and sequence homology with human PDZ domain-containing proteins involved in epithelial tight junction maintenance, including those from the MAGUK  and LAP (SCRIB) families.We have discovered through bioinformatics analysis that CPAF contains what represents a cryptic PDZ domain within the N-terminal subunit of the enzyme, spanning residues 106–212. When compared to canonical exemplary domains from each major PDZ class, CPAFCPAF is a secreted S41 protease in the chlamydial arsenal that recognizes a broad range of substrates within the active site. S41 proteases that exhibit broad active site substrate specificity will frequently utilize secondary specificity elements to restrict enzyme activation or catalysis to a defined subset of targets. This phenomenon is demonstrated by proteins in the tail-specific, DegP and HtrA protease families. In many of the S41 proteases, including DegP, HtrA2, HtrA3, and tail-specific proteases, secondary regulation of enzyme activity is achieved through communication between the catalytic serine protease domain and a nearby PDZ domain , 54, 55.106-212 is composed of a Tyr109-Leu110-Pro111-Tyr112 (YTPY). This YTPY sequence maintains the typical hydrophobic nature of the GLGF-loop despite the fact that the sequence is divergent from canonical PDZ domains  motif in the binding site loop for ligand recognition . The CPA domains .106-212 to the PDZ domains in human Dlg1, MPP7, and SCRIB revealed that Arg209 occupies an analogous region for the conserved basic residues from known PDZ domains  that occludes the protease domain active site [491, and CPAF106-212  substrate binding at the CPAF PDZ site induces conformational changes, similar to other S41 PDZ-containing proteases, after which the inhibitory sequence is presented to the active site in a catalytically competent conformation or (2) the singly cleaved inhibitory sequence obtains increased conformational flexibility, but remains bound to the CPAF PDZ domain to facilitate catalysis of the second and third cleavage events. Once the inhibitory segment is removed, the N- and C-terminal CPAF subunits associate to form the activated catalytic triad [This orientation of the inhibitory sequence argues for a possible role in simultaneously preventing ligand binding to the PDZ domain in addition to blocking substrates from reaching the protease active site. Removal of the inhibitory sequence is required for enzyme activity and may be facilitated by the PDZ domain. We propose that, if the CPAF PDZ domain is involved in zymogen activation, one of several outcomes may occur upon cleavage at the first activation site between Metic triad .172 and Ser176 may leave CPAF susceptible to hyperactivation as a result of activating PDZ ligands having easier access to the PDZ binding site.Interactions between the inhibitory helix and both the protease and PDZ domains highlight the potential for communication between the CPAF active site and PDZ domain. Such communication may form the basis for regulation of protease activation and/or substrate recognition. Distal regulation of protease activity is an established mechanism for the DegP and HtrA family hydrolases, as both classes exhibit PDZ-mediated oligomerization and activation , 40, 60.Following activation of the CPAF zymogen and formation of the active enzyme, substrates are either cleaved in discrete positions or completely degraded, further demonstrating the broad activity of the enzyme. This catalytic duality suggests that CPAF may operate as both an endo- and exo-peptidase. These different modes of cleavage may be the result of active site pocket selectivity or may be linked to contributions from secondary substrate specificity binding sites. The dichotomy of CPAF proteolysis mechanisms and the lack of a rigid active site consensus motif suggest that CPAF targeting may be dictated by distal regulatory elements, one of which may be the putative, N-terminal PDZ domain.E. coli tail-specific protease was shown to utilize a PDZ domain to assist in substrate binding. Mutations to the PDZ binding site resulted in a higher KM and lower kcat/KM for a peptide substrate [The concept of PDZ domains as secondary substrate recognition sites in proteases is well established. The HtrA and DegP proteases utilize the PDZ domains to form higher order oligomers and as secondary binding sites for substrates . In partubstrate . Chlamydubstrate .106-212 closely resembles human PDZ domains, we speculate that it may have evolved to hijack host protein-protein interactions, exposing them to potential CPAF-mediated proteolysis or directing CPAF to specific locales within the host cell. Alternatively, CPAF106-212 may intercept endogenous host PDZ ligands or PDZ-containing proteins to aid in molecular targeting of substrates. Others have shown that CPAF is released from the inclusion in late stages of infection [Because CPAFnfection , where iChlamydia infection disrupts tight junctions and adherens junctions in the single columnar layer of epithelial cells in genital tract and ocular infections, similarly to the sexually transmitted pathogen, N. gonorrhoeae [Many viral and bacterial pathogens have established a precedent of utilizing host-like PDZ domains or PDZ ligands as decoy pathogenesis effectors , 22, 26.orrhoeae .106-212 has been shown to be similar to human Dlg, MPPx, MAG1, and SCRIB, PDZ-containing proteins, which are known to be components of cell polarity and tight junction assembly in epithelial cells. MPP7, for example, co-localizes in epithelial cells with tight junction proteins Dlg1, E-cadherin, occludin and CASK, and builds assemblies at the interface of the outer membrane that act as scaffolding for formation of tight junctions [N. gonorrhoeae [106-212 to known PDZ-containing junction proteins indicates that CPAF may be involved in disruption of epithelial cell junctions to promote chlamydial infection.CPAFunctions . Chlamydunctions  and keraunctions , as wellunctions . Such a orrhoeae . Comprom106-212 may facilitate localization of the enzyme to interact with vimentin, vimentin binding proteins, or associated cytoskeletal filaments. Vimentin has also been implicated as a component of epithelial tight junctions and may be a cellular target for CPAF proteolysis late in chlamydial infections [While CPAF may directly intercept host PDZ-mediated signaling, it may also indirectly influence additional PDZ-associated proteins. CPAF has been postulated to degrade nectin-1, a protein required for assembly of adherens junctions in epithelial cells . We, andfections . FurtherMus musculus and Rattus norvegitus were detected through our bioinformatic searches. The CPAF protein sequences between C. trachomatis and C. muridarum are highly conserved, suggesting that the C. muridarum may contain a homologous PDZ domain Click here for additional data file.S2 Fig106-212 against 170 candidate PDZ domains are shown with the E-value and bit score cutoffs shown in blue and red, respectively. Any results with E-value greater than 1 were deemed insignificant and removed from analysis. In the inset plot, the concentration of data points in the left, upper quadrant indicates a population of homologous sequences that exceed the cutoffs for both alignment parameters.The E-values and bit scores for the results of the NCBI-BLAST alignment of CPAF(TIF)Click here for additional data file.


BRCA1 BRCT domains function as phosphoprotein-binding modules for recognition of the phosphorylated protein-sequence motif pSXXF. While the motif interaction interface provides strong anchor points for binding, protein regions outside the motif have recently been found to be important for binding affinity. In this review, we compare the available structural data for BRCA1 BRCT domains in complex with phosphopeptides in order to gain a more complete understanding of the interaction between phosphopeptides and BRCA1-BRCT domains. Eukaryotes have evolved to host sophisticated cell-cycle-dependent regulation networks to deal with DNA damage arising from both exogenous and endogenous sources. Dynamic protein–protein interactions and protein complex assemblies in the signalling cascades mediating DNA damage response (DDR) are often initiated by protein post-translational modification such as phosphorylation. Reversible interaction interfaces are created by phosphorylation on serine/threonine residues by key DDR-signalling-cascade regulators that include phosphoinositide 3-kinase-related kinases (PIKKs)   and checBRCA1 (breast cancer susceptibility gene 1) were first identified in patients with hereditary breast and ovarian cancer (HBOC)  domain  . Further) domain . Even th) domain , where B) domain  and poly) domain .A typical BRCT domain comprises between 90 and 100 amino-acid residues and folds as a globular domain with secondary structural elements ordered as βαββαβα. The first crystal structure of a BRCT domain, that of XRCC1, defined a four-stranded parallel β-sheet surrounded by three α-helices, in which helices α1 and α3 locate on one side of the β-sheet and α2 on the other side (PDB: 1CDZ)  Fig. 1aaFig. 1a.2The availability of protein structure data for BRCA1 tandem BRCT domains alone and in complex with various phosphopeptides has enabled us to understand and compare the protein-peptide interaction in detail. We first describe these interactions using a “2D interaction map” method, built upon the structural interaction fingerprint methodology used in the CREDO structural interactomics database  Fig. 2)Fig. 2). 3The 3D structures of BRCA1-tandem-BRCT domains show that the two BRCT domains (BRCT1 and BRCT2) are associated in a head-to-tail manner . A large3.1in vitro and in vivo assays have shown that M1775R mutant failed to bind to the BRCT domains  mediates polar interactions with evolutionarily conserved residues S1655/G1656 in β1 and K1702 in α2 of BRCA1 b Top. Th domains . Compari domains . The M17 domains .4Apart from the conserved “two-anchor” interaction sites, affinity differences among phosphopeptides towards BRCA1 BRCT domains can arise from the variation of the X residues at positions +1 and +2 in the pSXXF motif. Superposition of four peptides  bound to the BRCT domains shows the variation of residues at positions +1 and +2 c. The suResidues Gln, Thr and Val are found at the +2 position c. As exp5The N- and C-terminal sequences of the peptide outside the motif also contribute towards the peptide interaction. Phosphopeptides with the same core DpSPVF sequence and peptide length, but different N-terminal and C-terminal sequences, bind to BRCA1 BRCT domains with dramatically different affinities . In all The N-terminal region of the phosphopeptide contains more hydrophobic interactions with BRCT compared to the C-terminal sequence . SurfaceAside from the sequence differences, phosphorylation variants in the N-terminal phosphopeptide sequence can also occur. In addition to the phosphorylation at the Ser residue in pSXXF motif, an extra Ser residue in the N-terminal region can be phosphorylated for Bach1 and Abraxas . The str6Initial analyses of phosphopeptide binding to the BRCA1-BRCT domains focused on the pSXXF motif . As moreOur current understanding of the similarities and differences of structural interactions among these phosphopeptides bound to BRCA1-BRCT domains with respect to affinity underlines the fact that the phosphopeptides used for binding-affinity assays and structure determination usually represent only small parts of the protein structure. It is clear that we need to ask whether the rest of the phosphoprotein also contributes towards the binding with BRCA1. Understanding these detailed structures and binding modes of BRCA1 BRCT domains with various phosphopeptides can help us to understand the function of BRCA1 in various pathways and also contribute towards knowledge-based design and discovery of higher affinity BRCA1-BRCT inhibitors in the future.


Considering nearly 40% of multidomain proteins contain one or more discontinuous domains, we have developed DomEx to enable domain boundary predictors to detect discontinuous domains by assembling the continuous domain segments. Discontinuous domains are predicted by matching the sequence profile of concatenated continuous domain segments with the profiles from a single-domain library derived from SCOP and CATH, and Pfam. Then the matches are filtered by similarity to library templates, a symmetric index score and a profile-profile alignment score. DomEx recalled 32.3% discontinuous domains with 86.5% precision when tested on 97 non-homologous protein chains containing 58 continuous and 99 discontinuous domains, in which the predicted domain segments are within ±20 residues of the boundary definitions in CATH 3.5. Compared with our recently developed predictor, ThreaDom, which is the state-of-the-art tool to detect discontinuous-domains, DomEx recalled 26.7% discontinuous domains with 72.7% precision in a benchmark with 29 discontinuous-domain chains, where ThreaDom failed to predict any discontinuous domains. Furthermore, combined with ThreaDom, the method ranked number one among 10 predictors. The source code and datasets are available at  Proteins consist of one or several stable, compact, and autonomously folding substructures, which are referred to as domains. The identification of protein domains plays an important role in determining protein structures by experimental methods including Nuclear Magnetic Resonance (NMR) and X-ray crystallography,2. MeanwAlthough protein domains usually have a single continuous segment of protein chain, there are still many domains formed from two or more nonsequential segments, which are called “discontinuous domains”. For exaOver the last three decades, a number of methods have been developed to identify protein domains, which are roughly classified into two categories according to their input data: structure or sequence. The structure-based methods can accurately identify continuous and discontinuous domains from the atomic coordinates of proteins ,11–15. TAn accurate discontinuous domain prediction includes predicting the accurate domain boundaries and the number of segments within one discontinuous domain. Currently, the sequence-based methods mainly focused on domain number and boundary prediction. DGS guesses Some methods such as SnapDRAGON, RosettaIn this work, we present a new strategy, DomEx, to enable continuous domain boundary predictors to predict discontinuous domains based on the sequence segment assembly. A template similarity score, symmetric index score and a profile-profile alignment score were developed to detect the discontinuous domains through a comprehensive single-domain library collecting not only from the structure domain databases  from N- to C-terminal, and it has a homologous domain partner B. The position of the last residue of segment A1 is marked as n, then the alignment pair (A-B) could be divided into two segment pairs (A1-B1 and A2-B2) at the position between n and n+1. The coverage and sequence similarity of the segment pair A1-B1 should be close to that of the segment pair A2-B2. In other words, the coverage and sequence similarity between the two segment pairs are symmetric at the separated point.DomEx makes three assumptions: (a) Homologous protein domains can be detected by sequence-based profile-profile alignments; (b) Homologous domain pairs have approximately similar length; (c) The coverage and sequence similarity between the different segment pairs in the same homologous domain pairs are usually symmetric. Assumptions (a) and (b) are straightforward. For assumption (c), let some discontinuous domain A has two segments  score.Step 6: Detect conflicts and report the final result.The entire flowchart of DomEx is shown in Qi is predicted as discontinuous, if there is at least one hit Tj with length error e< 0.2, Ts-score>TTS and SI<TSI. The parameters TTS and TSI are the cutoffs of TS-score and Symmetric Index, and they satisfy the constraint functionTSI = f. This function can be decided by maximizing the Matthews Correlation Coefficient (MCC) value in the training datasets (see below).An assembled domain sequence If there are multiple candidates that pass through the decision tree, then the candidate with the lowest PPA-score is selected.X which is divided into n+1 segments  by n boundary bars , which can be predicted by ThreaDom  and , respectively. The average MCC, recall and precision for the training dataset with different TTS and TSI are shown in An exhaustive grid search of the 2-dimensional space of TS-score and SI was performed to find the best combination of cutoffs The Symmetric Index becomes more important when TS-score <0.5. The highest MCC values (> 0.3) are in the region of TS-score ∈ and SI∈, where DomEx has a reasonable precision (>0.6) and maximum recall (>0.34).TSI = f with a dynamic relationship between the cutoff TTS and the cutoff TSI controlled by a single parameter b, i.e.TTS = 0.30, and we get TSI = 0.2 according to the TSI = 0.30 and SI≤TSI = 0.2. Algorithm 2 . The PPA-score gives the alignment quality between the query and template sequence. The score is usually negative. A low score means a good alignment. In the ECV training, there are 109 out of the 288 positive samples correctly detected as discontinuous, and 72 out of the 548 negative samples incorrectly detected as discontinuous when b>0.1. We use a total of 181 alignments as input to train the best PPA-score cutoff. We found the PPA can improve the MCC and precision when b<0.5 and PPA-score <-1.90 when the templates are collected from Pfam. Then the query is treated as a “Positive” detection, when the parameter b<0.5 and the PPA-score <-1.90.TPPA<−1.90 as the default parameters in DomEx.We tested the method on 390 discontinuous-domain samples on the Validation Dataset, using b from 0.9 to 0.1 with step -0.1 and PPA-score <-1.90 when b<0.5. Similar to the tendency on the Training Dataset, the precision of DomEx in the Validation Dataset varies from 1.0 to 0.771. The MCC values are all higher than 0.30 with recall ranging from 0.183 to 0.339. For better balance between precision and recall, we selected b = 0.3 and PPA-score cutoff To detect the discontinuous domains, DomEx depends on the predicted boundaries from other predictors, such as ThreaDom. The test dataset TEST-SET-I contains 99 positive and 58 negative discontinuous domains which were derived from a non-redundant set of protein chains consisting of 97 discontinuous domains . Each boundary was predicted by ThreaDom within an error of ±20 residues to the boundary definitions in CATH 3.5.In As a control, we employed five publicly available domain predictors, including ThreaDom, FIEFDomTo test the situation where there are only weakly homologous templates, for ThreaDom, we excluded all templates that have a sequence identity >30% to the target protein, and we also excluded templates that are detectable by PSI-BLAST with an E-value<0.05.For FIEFDom, we kept two group boundary prediction results, FIEFDom I and FIEFDom II. FIEFDom I excludes the templates if their sequence identity >30%; while FIEFDom II includes all the templates.The dataset TEST-SET-II contains 481 multi-domain chains, which is the same size as the training and validation sets. It includes 326 discontinuous chains and 155 continuous domain chains. Each chain has at least 3 segments, and the length of each segment is not less than 40 residues. When detecting the discontinuous domains, both DomEx with ThreaDom boundaries (ThrDm_Bdr+ DomEx) and DomEx with FIEFDom II boundaries (FIEFDom II+DomEx) have a higher NDO-score than their boundary prediction without DomEx detection. And the NDO-score of ThrDm_Bdr+DomEx (0.70) is higher than all the predictors that do not support discontinuous-domain detection. ThreaDom+DomEx has the highest NDO-score of 0.759. The results demonstrate that DomEx can improve the discontinuous domain detection when combined with other boundary predictors because of the addition of a sequence-based domain library and the symmetric alignment score.There are a total of 17 targets which have > = 3 continuous segments (length > 30 residues) from CASP8 to CASP10 experiments. Six contain multiple continuous domains, and eleven contain at least a discontinuous domain. A summary of the domain definitions from the CASP assessors is listed in b>0.8), DomEx achieves a high precision and is independent of the template sources (CATH+SCOP or Pfam). When b<0.8, the accuracy of the Pfam-based prediction is significantly lower than CATH+SCOP. If the template comes from CATH or SCOP, DomEx has a high precision (>90%). If the templates come from Pfam, the precision declines to about 50% when b is less than 0.65.The domain sequence library of DomEx is based on CATH, SCOP and Pfam-A. The domain boundaries of SCOP and CATH are defined based on the 3D structure of the proteins. Pfam-A is based on the HMM classification of sequences from whole-genomes. It is observed that the accuracy of templates from the Pfam library is about 60~70% of that from CATH+SCOP. b. For large b, the templates come mainly from CATH and SCOP, while for small b, much more templates are from Pfam.  When b = 0.2, about 70% of the templates come from Pfam, and the recall of DomEx is about 44%. The recall is 100% higher than when b = 0.5, where 50% of the templates come from Pfam. The templates from Pfam increase the recall of DomEx, even though the precision of DomEx decreases when b decreases. The domain sequences from Pfam improve recall because of its large size relative to the other databases.TPPA in b when TPPA varies from 0.0 to -5.0 with step -0.1. The average MCC and precision of Group I, which are 0.341 and 0.901, respectively, are higher than the other two. From the table, one can see that Group I has higher recall and MCC values for the cases when the precision values are similar to the other groups (underlined in table). For example, Group I has precision of 80.6% at b = 0.3, which is a bit higher than Group II at b = 0.4 and Group I at b = 0.5, while its recall is 31% and 61% higher than the other two, respectively.In DomEx, the Profile-Profile Alignment combined with the predicted secondary structure information is used to help DomEx improve the low precision when the templates are from Pfam. PPA filtering is not as effective for Group II as it is for Group I. The main reason is the domain definitions from SCOP and CATH are more likely to be correct than Pfam since they are derived from known 3D structures. Therefore, we used PPA filtering only for templates from Pfam library.In the segment assembly, we considered only the segment orderings from N- to C-termini, e.g. (A1A2) in Nevertheless, the reversed segment assembly can detect some interesting domain structures. All the three reversed cases are from proteins with segment-swapping domains (SSDs) . There aWe have proposed a new strategy, DomEx, to extend the ability of domain boundary predictors to detect discontinuous domains. The method assembles and detects discontinuous domains from the sequence segments. DomEx incorporates template similarity, symmetry of segment pairs, profile-profile alignments, and structure-based and structure-free libraries.https://github.com/xuezhidong/DomEx.Two test benchmarks showed that DomEx not only worked with the boundary predictors, but also was complementary to the discontinuous-domain detection method in ThreaDom. DomEx recalled 26.7% of the cases where ThreaDom failed. Half of these cases are attributed to templates from Pfam. When compared to other methods, DomEx plus ThreaDom gave the best NDO score, which further confirms that DomEx can detect discontinuous domains even without known 3D-structure templates. The main advantage of DomEx is that it searches for templates using domain-domain alignments rather than chain-chain alignments. Using domain-domain alignments improves recall because chain-chain alignments may miss templates where the domains match, but the rest of the chain does not match well. The benchmark results show that DomEx is an effective method, which opens the possibility of finding discontinuous domains in genome-wide studies. Currently, DomEx supports the detection of two-segment discontinuous domains. Further work will extend the model to detect discontinuous domains with more than 2 segments, and try to utilize the domain annotated information from CATH, SCOP and Pfam to enhance the performance of detecting discontinuous domains. The accuracy of boundary predictors and sequence alignment tools should also improve the detection results. The source code and datasets of DomEx are available at S1 Table(PDF)Click here for additional data file.


The crystal structure of PvuRts1I was determined and a 5-hydroxymethylcytosine-binding pocket was identified in the SRA-like domain. Enzyme variants were engineered to assist in hydroxymethylome mapping based on the crystal structure of PvuRts1I. 5-Hydroxymethylation is a curious modification of cytosine that was discovered some decades ago, but its functional role in eukaryotes still awaits elucidation. 5-Hydroxymethylcytosine is an epigenetic marker that is crucial for multiple biological processes. The profile is altered under certain disease conditions such as cancer, Huntington’s disease and Alzheimer’s disease. Using the DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), the hydroxymethylome can be deciphered at the resolution of individual bases. The method is based on the enzymatic properties of AbaSI, a member of the PvuRts1I family of endonucleases. PvuRts1I is a modification-dependent endonuclease with high selectivity for 5-hydroxymethylcytosine over 5-methylcytosine and cytosine. In this study, the crystal structure of PvuRts1I was determined in order to understand and improve the substrate selectivity. A nuclease domain and an SRA-like domain are located at the N- and C-termini, respectively. Through comparison with other SRA-domain structures, the SRA-like domain was proposed to be the 5-hmC recognition module. Several mutants of PvuRts1I with enzymatic activity restricted to 5-hydroxymethylcytosine only were generated based on the structural analysis, and these enzyme variants are appropriate for separating the hydroxymethylome from the wider methylome. E. coli Rosetta2 (DE3) cells (Novagen) carrying the vector were grown in LB medium at 310 K to an OD600 of 0.6–0.8 and were then induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 20 h at 289 K. Cells were harvested by centrifugation and lysates were prepared by sonication in lysis buffer , cleared by centrifugation and applied onto a nickel–nitrilotriacetic acid (Ni–NTA) Superflow column (Qiagen) pre-equilibrated with lysis buffer. Washing and elution were performed with lysis buffer containing 25 and 250 mM imidazole, respectively. The eluted proteins were applied onto a HiLoad 16/60 Superdex 200 gel-filtration column  in buffer A . Purified protein was concentrated to 6 mg ml−1 for crystallization. Mutations of PvuRts1I were generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the target sites. Mutant proteins were expressed and purified under identical conditions to those used for wild-type PvuRts1I.The sequence encoding PvuRts1I was synthesized using the optimized E. coli Rosetta2 (DE3) cells in M9 medium supplemented with selenomethionine (Sigma–Aldrich) at a final concentration of 60 mg l−1 using the methionine-biosynthesis inhibition method. Se-PvuRts1I was purified identically to native PvuRts1I.Selenomethionine-labelled PvuRts1I (Se-PvuRts1I) was expressed in 2.2.−1) was mixed with 1 µl of a well solution consisting of 1.13 M NaH2PO4, 0.75 M K2HPO4, 0.1 M CAPS pH 10.5, 0.19 M Li2SO4, 6%(v/v) glycerol. The mixture was equilibrated against 100 µl well solution at 286 K. Single crystals were obtained after 4 d.Crystals of Se-PvuRts1I were obtained using the sitting-drop vapour-diffusion method. 1 µl Se-PvuRts1I (6 mg mlv/v) glycerol and flash-cooled in liquid nitrogen. X-ray diffraction data were collected on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility. Diffraction data for Se-PvuRts1I crystals were processed using MOSFLM, POINTLESS and SCALA from the CCP4 suite . The quality of the final model was validated using PROCHECK  was mixed with 1 µl PvuRts1I or the particular mutant (1 µg µl−1) and 2 µl NEB buffer 4  in a 20 µl final volume. The mixture was incubated for 1 h at 23°C and then resolved on a 1% agarose gel.The PCR products were purified using the TIANquick Midi Purification Kit (Tiangen Biotech). For assessment of enzyme activity , 10 µl substrate DNA , 100 ng substrate DNA was digested by PvuRts1I or an enzyme variant in a twofold serial dilution in NEB buffer 4. The protein concentration of the sample applied to lane 1 was 4 mg ml3.3.1.a). The PvuRts1I crystals diffracted to 2.9 Å resolution and phases were obtained by the single-wavelength anomalous dispersion (SAD) method. X-ray diffraction data-collection and structure-refinement statistics are shown in Table 1To investigate the substrate specificity of the enzymes belonging to the PvuRts1I family, we solved the structure of full-length PvuRts1I. Although PvuRts1I was crystallized in the presence or absence of a 29 bp DNA fragment, we could only obtain crystals without substrate bound Fig. 1a. The Pa and 1b). The endonuclease domain is located in the N-terminal part and the DNA-binding domain constitutes the C-terminal portion . The two domains are connected by an irregular secondary structure which consists of a short α-helix (α5) and two β-strands packed against each other (β6 and β15). The N-terminal endonuclease domain adopts a typical three-layered α–β–α sandwich architecture, with a central five-stranded β-sheet flanked by three α-helices on one side and one α-helix on the other side . The C-terminal DNA-binding domain contains eight β-strands and one α-helix, giving an overall β-barrel-like structure with one side open. The concave surface of the open side houses the DNA-binding site .The structure of PvuRts1I consists of six α-helices and 15 β-strands, which fold into two distinct domains .A search of the protein-structure database using DALI server revealed that the C-terminal DNA-binding domain of PvuRts1I is related to 5-mC/5-hmC binding modules, including the SRA domains of Arabidopsis SUVH5  and human UHRF1  and the SRA-like domain of another modification-dependent restriction endonuclease MspJI . Therefore, the DNA-binding domain of PvuRts1I is referred to as the SRA-like domain. As for other 5-mC/5-hmC binding modules, the overall shape of the SRA-like domain of PvuRts1I resembles a saddle, with a concave surface at the open side of the β-barrel. In other SRA or SRA-like domains the binding site of the modified cytosine is located on this concave surface. Similarly, we speculate that PvuRts1I recognizes 5-hmC using this surface.Comparison with structures in the PDB using the 3.2.a), which is consistent with a previous report . In addition, this dimer is also identified by the PISA server . The buried surface area of the interface is approximately 812 Å2, which is clearly adequate to stabilize the dimer in solution. This observation supports the idea that a dimer is the functional unit of PvuRts1I family endonucleases . However, the NKR finger loop of PvuRts1I is much shorter than the equivalent loops of SUVH5 and UHRF1, suggesting that PvuRts1I might recognize the substrate DNA through the thumb loop.As described above, the SRA-like domain of PvuRts1I is assumed to be the substrate-binding module. To shed light on the mechanism of substrate recognition, this domain was compared with those of other modified cytosine-binding modules . Although already narrow and deep in the case of ligand-free human UHRF1 , this pocket becomes narrower and deeper still upon binding 5-mC owing to a quite dramatic conformational change . At one end of the pocket there are two glycine residues that are potentially responsible for the conformational change owing to the intrinsic flexibility associated with this amino acid . Similarly, the SUVH5 5-mC binding pocket is also narrow and deep  and contains an equivalent pair of glycines . In PvuRts1I, Tyr210 and Ala212 are found in place of these glycines, which are much more rigid than glycine . As a result, the binding pocket of PvuRts1I may not be able to undergo this conformational change . Sequence alignment shows that Tyr210 is conserved in the PvuRts1I homologues, indicating that it might contribute to substrate selectivity .In the SRA domains of SUVH5 and human UHRF1 in complex with DNA, the 5-mC base is flipped out of the DNA duplex and inserted into a binding pocket . Interestingly, two tryptophan residues (Trp205 and Trp215) are located at equivalent positions in PvuRts1I, which provide aromatic stacking interactions with the 5-hmC of the substrate DNA . In addition, the side chains of Asp469 in UHRF1 and Asp418 in SUVH5 form hydrogen bonds to N3 and N4 of 5-mC, respectively. In PvuRts1I, Asn217 is situated at the same location, suggesting that this residue may perform a similar functional role . Furthermore, the main-chain carbonyl groups of Thr478 in human UHRF1 and Thr429 of SUVH5 make further hydrogen bonds to N4 of 5-mC. In PvuRts1I, Glu228 is positioned in this vicinity and may be involved in equivalent interactions . Sequence alignment of PvuRts1I homologues from different species indicates that the amino-acid residues proposed to interact with the modified cytosine are highly conserved  and therefore are likely to contribute to the substrate selectivity.In both the human UHRF1 and SUVH5 SRA–DNA structures, the 5-mC base is sandwiched by π-stacking interactions between two aromatic amino acids: Typ466 and Tyr478 or Tyr416 and Tyr428, respectively , presumably owing to the involvement of these residues in stabilizing the cytosine base in substrate DNA. Intriguingly, mutating Tyr210 to Phe and Ala212 to Asn exhibited opposite effects on substrate selectivity, despite their close proximity in the sequence. The preference of the the A212N mutant for 5-hmC over 5-mC and cytosine decreased significantly compared with the wild-type enzyme, while the selectivity of the Y210F variant increased . Although the endonuclease activity of the N217K mutant was completely abrogated, the selectivity towards 5-hmC of the N217A mutant was enhanced . Strikingly, the N217D mutant was found to retain similar endonuclease activity towards 5-hmC as wild-type PvuRts1I, while its activity towards 5-mC and cytosine was lost .Since wild-type PvuRts1I assembles into a functional dimer, we first examined the assembly of these mutants to confirm their correct oligomerization, which is probably required for activity. Fortunately, none of the mutations prevented the formation of a dimer in solution . The endonuclease activities of the W215A and E228K mutants were completely abolished Fig. 5b, presued Fig. 5b. Althoed Fig. 5b. Strikst Fig. 5b.et al., 2011a). For the Y210F mutant this was 256:8:ND  and for the N217D mutant this was 32:1:1 . Specificity towards 5-hmC was increased in the Y210F and N217D mutants . The Y210F and N217D mutants are therefore probable candidates for distinguishing between 5-hmC and 5-mC.In order to further quantify the relative selectivity of PvuRts1I and its variants towards different cytosine modifications, we first investigated the appropriate reaction time for detecting relative selectivity. This showed that 5-hmC is digested in less than 10 min . However, in order to measure the relative selectivity of different modified cytosines, 60 min is a more appropriate reaction time. We then applied the same approach as previously used for PvuRts1I family enzymes , apart from the N-terminal tag which was not cleaved in their work and two fragments (between residues 71 and 76 and between residues 181 and 184) which are disordered in our structure.In order to uncover the molecular mechanism underlying the relative selectivity towards 5-hmC, 5-mC and cytosine, we solved the crystal structure of PvuRts1I. The structure consists of two domains: a novel SRA-like domain that recognizes 5-hmC and an unusual nuclease domain that has not previously been curated by the NCBI Conserved Domain Database . The distance between the putative binding sites of the two SRA-like domains is 75 Å . It has previously been shown that the recognition site of PvuRts1I is 5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′ . In addition, comparison of the UHRF1 SRA domain in the presence or absence of substrate DNA revealed a large conformational change in the 5-mC binding pocket after insertion of 5-mC. Two glycine residues on one edge of the binding pocket may be responsible for this induced-fit mechanism of 5-mC recognition. In PvuRts1I, Tyr210 and Ala212 replace these glycine residues, which results in a more rigid pocket of fixed shape and size . The crystal structure of PvuRts1I bound to the 5-hmC DNA substrate is needed to unambiguously clarify the mechanism of substrate recognition.In prokaryotes, there are two characterized proteins that can bind hydroxymethylated DNA, namely McrB and MspJI . Why Tyr210 plays such a key role in the recognition of 5-hmC remains unknown at present. A crystal structure of PvuRts1I bound to its 5-hmC DNA substrate would be highly informative in order to clarify the substrate-selectivity determinants.In a recent report, AbaSI was used to map the genomic distribution of 5-hmC (Sun In summary, the crystal structure of PvuRts1I determined in this study, coupled with structural and biochemical analysis, allowed us to engineer enzyme variants that may perform better than AbaSI in the Aba-seq method and may assist in hydroxymethylome mapping and future 5-hmC research.PvuRts1I, 4okyPDB reference: 10.1107/S139900471401606X/qh5007sup1.pdfSupporting Information.. DOI:


How is higher cognitive function organized in the human parietal cortex? A century of neuropsychology and 30 years of functional neuroimaging has implicated the parietal lobe in many different verbal and nonverbal cognitive domains. There is little clarity, however, on how these functions are organized, that is, where do these functions coalesce  and where do they divide . Until now, there has been no multi-domain synthesis in order to reveal where there is fusion or fission of functions in the parietal cortex. This aim was achieved through a large-scale activation likelihood estimation (ALE) analysis of 386 studies (3952 activation peaks) covering 8 cognitive domains. A tripartite, domain-general neuroanatomical division and 5 principles of cognitive organization were established, and these are discussed with respect to a unified theory of parietal functional organization. A key goal of cognitive neuroscience is to relate cognitive functions and dysfunctions to underlying neural processing. A common approach, adopted in functional neuroimaging, cognitive neuropsychology and other related neuroscience methods, is to explore 1 specific behavioral domain  and to investigate which brain regions are implicated and how their function varies following changes to important task-related parameters. The considerable productivity of the field is such that significant progress has been made in dissecting both the cognitive machinery and the underlying neural networks in many of these specific behavioral domains. Despite, or perhaps because of this success, 3 key challenges are emerging: 1) like most other natural sciences in the twenty-first century, vast quantities of data are produced, increasing the need for meta-analytic tools to distill the core, reliable findings not only within each specific behavioral domain but also across them; this is because 2) if we step beyond the large literature dedicated to each behavioral domain, then it becomes readily apparent that many neural regions are common to numerous, diverse behavioral activities; generating the conclusion that 3) there is no simple one-to-one mapping between each type of activity and a single underlying brain region. Instead, each behavioral domain maps onto a cortical network, and in reverse, each cortical region seems to play a role in a variety of different cognitive behaviors. Indeed, this neuroscientific maelstrom can sometimes feel like a Shakespearean comedy in which a multitude of characters have a bewildering array of relationships and assume multiple roles. In order to avoid turning comedy to tragedy and simply treating this confusion as some kind of masterpiece, a necessary first step is to map out the overlapping neural bases of different behavioral domains “simultaneously” and then search for a unifying explanation.This investigation focused on the complexity of cognitive functions associated with the  parietal cortex. This region Fig.  can be sMany theories have been generated in relation to the potential neurocomputations of the parietal cortex. Most  of these hypotheses try to account for findings from 1 behavioral domain  and, therefore, tend to be domain specific in nature. Examples of these accounts include the “semantic hypothesis”  in whichOriginally highlighted in Henry Head's seminal work –posterior (AG) dimension. Supramarginal and angular gyri and AG show distinct patterns of connectivity  whereas “Positive versus negative activations”: Unlike most other cortical regions, VPC is associated with the DMN ; this re“Laterality”: A final potential dimension of parietal organization reflects left versus right hemispheric differences. Chronic disorders of language or apraxia are associated with left parietal damage whereas long-term visuospatial attention impairments  tend to follow from right parietal lesions . This paTo assess the principles of parietal organization, we undertook a large-scale, multi-domain meta-analysis based on the considerable functional neuroimaging data that are now available in the literature . We define “behavioral domains” here as a way of categorizing/differentiating different behaviors or higher mental activities . Each of these will be underpinned by a combination of domain-specific and domain-general neurocomputations. If parietal organization reflects a multitude of mutually exclusive domain-specific functions then the meta-analysis results should resemble a form of “neuromarquetry.” Alternatively, if a smaller set of domain-general neurocomputations underpins a wide range of behaviors then the pattern of activations should coalesce into a shared region . Briefly, we applied activation likelihood estimation (ALE) analysis . ALE is a coordinate-based meta-analysis technique that computes the activation likelihood for each voxel and compares this with a null-distribution to determine statistical significance. This method also allows direct comparison of ALE maps to determine statistically significant differences. As noted earlier, some domain-specific reviews have been previously reported. For formal direct comparisons across domains and to link the present unified meta-analysis directly back to the literature, studies were re-selected from key reviews .This investigation was based, therefore, on a multi-domain synthesis of the considerable functional neuroimaging data that are now available. Through this large-scale meta-analysis, the behavioral–neuroanatomical relationships within the parietal cortex (as well as other regions) were mapped in order to establish 1) which behavioral activities coalesce (indicating shared underlying neurocomputation) and 2) which neuroanatomically dissociate . The results were used to test 5 potential organizational principles that emerge from the literature (summarized later) as well as specific theories.analysis  to 3952  reviews  and suppSupplementary Material) (http://www.brainmap.org/). Specifically, we selected all studies of healthy participants  in the database that showed task-related deactivations relative to a low-level control condition (either fixation or rest). This is considered a robust and unbiased method for identifying the DMN and has been used successfully elsewhere in the literature .Studies were selected from the behavioral domains of episodic retrieval, semantic cognition (separating automatic semantic activation and “top-down” semantic-based decisions), phonological processing, sentence-level processing, numerical tasks , tool-related tasks, attention (separating top-down attention and bottom-up attention), and the DMN. This is not an exhaustive list of all behavioral domains that have been associated with the parietal cortex, but they do represent a diverse range of behavioral activities for mapping parietal functions . Some other behavioral domains could not be included due to an insufficient number of studies for the ALE method. We aimed for a representative sample of the literature from each domain and, in particular, for the current multi-domain meta-analysis to link directly back to previous seminal reviews or meta-analyses of individual behavioral domains. To this end, studies were re-selected from those included in key reviews or meta-analyses of each behavioral domain. In doing so, this multi-domain meta-analysis was able to preserve the selection criteria and other parameters that are considered highly relevant to researchers working in each subject area  . When reterature . OverallP < 0.05 and a minimum cluster size of 100 mm³. To determine the level of commonality across domains, we examined the overlap from the resultant ALE maps. Figure The ALE analyses were carried out using GingerAle 2.1 . To reveP < 0.05). Secondly, to test for domain-specific processes within dorsal or ventral areas, we directly contrasted each behavioral domain with one another, separately for dorsal and ventral tasks . Finally, we conducted laterality tests for each domain to investigate any significant differences in ALE values across hemispheres. This was achieved by contrasting each set of foci with that from an identical set but where the x coordinate had been inverted .Three subtraction analyses were carried out to verify that differences observed in the primary ALE analysis were statistically significant. Visual inspection of the results from the primary individual-domain ALE analyses suggested that there was a difference in the domains that engaged dorsal and ventral parietal areas. This dorsal–ventral difference is in agreement with the second organizational principle highlighted in Introduction. Specifically, we found that certain domains engaged DPC, whereas ventral areas were engaged by a different set of tasks other. Furthermore, within each area, recruitment was highly overlapping across domains but between areas there was no overlap. To test these apparent dorsal–ventral differences formally, the first subtraction analysis examined whether this difference was statistically significant by directly contrasting all “dorsal” tasks with all “ventral” tasks . These data were collated across foci for each task and neural region to examine any systematic patterns in the data.Next, we looked for systematic effects across tasks and neural regions in the tendency for positive or negative activation relative to baseline. Specifically, we noted the direction of relative activation levels (positive or negative) compared with rest/fixation for each foci contributing to the significant clusters in the primary ALE analysis . While the focus in the main paper is primarily on parietal activation, the non-parietal results are relevant to on-going debates about the roles of prefrontal cortex and posterior temporal regions in various behavioral domains . Thus, in the Supplementary Material, we also report results from within lateral prefrontal recruitment in linguistic tasks , and the recruitment of posterior temporal areas by top-down semantics and tool-related tasks.Foci from outside parietal cortex were also included in the ALE analysis . A subtraction analysis confirmed that DPC tasks were significantly more likely to engage bilateral IPS and SPL, whereas VPC tasks were significantly more likely to engage the left AG and precuneus .The primary ALE analysis revealed a clear dorsal–ventral distinction, with different task components modulating DPC and VPC areas. Within VPC, a further anterior–posterior difference (SMG vs. AG engagement) was apparent. The results are summarized in Figure Several tasks engaged a common IPS/SPL region including top-down attention, numerical calculation, executive semantic decisions, tool-praxis, and phonological decisions. ALE clusters were highly overlapping  with some minor variations in cluster extension , whereas for sentence-level tasks and episodic retrieval, activation was almost exclusively positive . Numerical fact retrieval also showed a tendency for positive activation . Interestingly, the tasks that have been contrasted most often with numerical fact retrieval  deactivate AG (100% of cases). In summary, despite the high neuroanatomical overlap across AG tasks, the polarity of AG activation varies dramatically. This novel fact suggests an important organization principle for the functional role of the AG versus other parietal regions.There were systematic differences in activation versus deactivation across tasks and spatial locations : 1) left-only for executive semantics, phonological processing, and tool-praxis decisions; 2) bilateral (left > right) for automatic semantic retrieval, episodic retrieval; 3) bilateral (left = right) was found for attention (bottom-up and top-down attention), numerical calculation, and sentence-level tasks.Three patterns were revealed by the ALE analysis  the extreme positions of either highly “fractionated” models or complete functional unification are unlikely to be correct. Instead, the tasks appear to coalesce into a triad of regions; 2) the fates of these areas are not governed by domain but rather the cognitive components required by each task. Given the diversity of tasks that engage the 3 same areas, an explanation based on domain-general processing systems seems the most potent.“Dorsal versus ventral parietal regions”: In keeping with classical neuropsychology and functional neuroimaging , the dat“Anterior versus posterior areas”: Within VPC, the data indicate a secondary split such that phonology and “bottom-up” attention are associated with ventral SMG/temporoparietal junction. The remaining tasks and DMN are linked to the AG. Thus, beyond the dorsal–ventral distinction, the current data suggest an additional anterior–posterior VPC gradient. This dimension was not considered in the original versions of the dual-processing model, but it has been highlighted by direct contrasts of attention and episodic memory  and has Dimensions 1–3 indicate the presence of 3 functionally dissociable, multi-domain parietal networks: IPS/SPL, AG and SMG. This finding fits with resting-state functional connectivity and tractography studies, in which the same areas demonstrate different connectivity patterns: The AG forms part of the DMN alongside the medial prefrontal cortex, posterior cingulate cortex, and medial temporal lobes; the SMG forms part of a cingulo-opercular system involving the lateral prefrontal cortex, anterior cingulate cortex, and frontal operculum/insula; and IPS/SPL is part of a fronto-parietal control system connected with dorsolateral and medial prefrontal cortex (supplementary motor area), MT+ and the frontal eye fields .“Activation polarity”: A new functional dimension was revealed by the meta-analysis. In IPL/SPL and SMG, activations were positive across all tasks (> rest/fixation). In contrast, the AG appears to exhibit a full spectrum from deactivation for the many tasks associated with deactivation in the DMN as well as “automatic” semantic processes, through to positive activation for sentence-level tasks, episodic and number-fact retrieval.“Laterality”: The data indicate graded differences across hemispheres which do not align neatly with a verbal versus nonverbal distinction. The only tasks to be associated with a left-only distribution were phonological, executive semantics and tool-praxis decisions. This is consistent with the observation that chronic  aphasia and apraxia are associated with left-hemisphere damage . The remWhat does this multi-domain meta-analysis tell us about the 5 potential principles of parietal organization?The extant theories vary in how well they map onto these 5 dimensions of parietal organization. Many of these hypotheses were created to explain various “domain-specific” results, including number, space and time ; semantiSeveral researchers have suggested that DPC forms part of a domain-general fronto-parietal executive control system . ConsistThe DPC results are less consistent with the strong “fractionated” view . It shouThe majority of VPC-associated tasks  are more automatic, stimulus-driven and have lower executive demands . This contrapositive relationship between DPC and VPC is consistent with the thrust of the domain-general, dual-attention hypothesis or the internal versus external processing hypothesis for the DMN  and is lMany domain-specific hypotheses have been formulated around processing in the AG or SMG. Thus, a second explanatory approach could be generated from the assumption that DPC supports a domain-general executive system whereas domain-specific processing occurs in VPC. No one domain-specific theory, however, appears to be able to explain the multi-dimensional counterpoint pattern of VPC data. Key challenges are found in explaining the overlap of functions in ventral SMG and the separate set in AG, as well as its negative-to-positive activation polarity. First, we need an explanation for why left vSMG acts simultaneously as a phonological buffer/sensory-motor interface for speech , and bilAn alternative approach suggests that either the AG supports temporary storage of multimodal episodic information, which acts as an interface between executive processes and stored episodic representations , or the “Principle 1—tripartite division”: follows directly from these 2 sets of assumptions. Multimodal buffering and emergent time/space/context statistical extraction are required across many different verbal and nonverbal activities and thus domain-general patterns emerge. The tripartite division emerges as a consequence of the different long-range connectivity patterns.“Principle 2—dorsal versus ventral”: By connecting with lateral prefrontal cortex, DPC forms the domain-general multi-demand network . Most noTo clarify our working hypothesis, we propose that parietal organization is not split by “domain”/task, rather it reflects the differing neurocomputations needed to meet the demands of every task. Thus, for the same task, different parts of the parietal cortex will be engaged and this pattern could change during a task. We propose that both VPC and DPC might act as a buffer of activated information but the precise neurocomputation varies depending on connectivity—resulting in dorsal areas acting as an executive buffer and ventral areas as an automatic buffer. These 2 networks are, however, closely inter-related. For example, during a well-rehearsed activity, VPC will automatically buffer the multimodal on-going information, which will be useful for timing and coordinating steps in sequential and multimodal activities. During the activity, however, if a stimulus or string of stimuli enter the automatic buffer that have high valence or violate expectations , then this will trigger a saliency signal, which in turn would activate top-down processes. Thus, in this situation, the same task will engage both VPC and DPC depending on the demands of the situation. This is akin to and inspired in part by the notion of a “circuit-breaker” proposed by “Principle 3—anterior versus posterior”: VPC subdivisions probably also emerge from differential connectivity of SMG versus AG to various temporal and frontoinsular regions . AG invo“Principle 4—polarity of activation”: Taking each region in turn: 1) as a part of the multi-demand system, DPC activity will rise in line with task difficulty, 2) VPC will activate when tasks tap into long-term, buffer-extracted, content-invariant statistical structures , but for executively demanding tasks that do not require this information, continued automatic buffering of related and unrelated, external events in VPC is disruptive to targeted, goal-directed decisions supported by DPC. Performance can be improved, therefore, by inhibiting the VPC “slave” buffering  but, in order to balance task performance against awareness to on-going external events, VPC inhibition is titrated against task difficulty, thus explaining why VPC deactivation is greatest for the most challenging tasks or stimuli.“Principle 5—laterality”: Most tasks recruit DPC/VPC bilaterally except phonology, semantics, and object praxis. Thus, a verbal/nonverbal distinction cannot underpin the limited laterality differences. Instead, the left-sided pattern for phonology and praxis could reflect connectivity to prefrontal motor control areas which, for speech and fine-motor movements, is strongly left-lateralized  whereas This penultimate section sketches out a new unified framework that attempts to place a girdle around the 5 dimensions of parietal organization. It adopts existing ideas from the domain-general and domain-specific theories and assimilates them with insights from connectivity information and computational modeling. Parietal unified connectivity-biased computation (PUCC) is founded on 2 sets of assumptions. The first is that ventral brain pathways extract information about content  that can be generalized across time and context , whereasAs noted in the section Materials and Methods, this overarching meta-analysis did not contain an exhaustive coverage of all cognitive domains that engage parietal cortex. Some of these have an insufficient number of studies for ALE analysis, but 2 domains have been carefully studied previously (working memory and theory of mind) and these independent meta-analyses provide further support for the PUCC hypothesis. A meta-analysis of working memory studies (n-back tasks) showed that DPC is reliably activated , the locAlthough this discussion has inevitably focused primarily upon the functional neuroimaging data, we finish by considering how the PUCC framework fits with the large and long-standing neuropsychological literature. Indeed, as noted earlier, observations from classical and contemporary neuropsychological studies were part of the motivation for this unified meta-analysis and have inspired some of the principles. Although it is only relatively recently that functional neuroimaging studies have explored the potential parietal overlap across different behavioral domains, this notion has a long history in neuropsychology; Bálint's and Gerstmann's syndromes, for example, were denoted as “syndromes” because the same underlying lesion caused a constellation of co-occurring symptoms across a range of tasks/behavioral domains . With regard to Principle 2 , again the neuropsychological findings would appear to be consistent with the general patterns found in this unified meta-analysis. For example, Bálint's syndrome (resulting from DPC damage) is associated with impaired object-directed reaching (optic ataxia) and intentional eye-movements (oculomotor apraxia) i.e., tasks that are goal-directed. Contemporary lesion-based neuropsychology has also highlighted that DPC damage is associated with working memory  deficits rather than simple span or recall ability  FurthermThe separation of SMG and AG functions (Principle 3) is fairly well-established in the neuropsychological literature. As noted in Introduction, within the verbal domain, phonological deficits are associated with lesions to or stimulation of SMG  whereas Finally, Principle 5 fits fairly readily and consistently with the neuropsychological literature. As noted in Introduction, chronic aphasia and apraxia are associated with left-hemisphere lesions and these behavioral domains exhibited a left-sided distribution of imaging peaks in this unified meta-analysis. Other domains appear to be bilateral in nature, and this predicts that clear, clinically notable neuropsychological deficits should be associated with biparietal diseases . One poAs a final postscript to this neuropsychological discussion, we should note that there are intrinsic challenges in relating the neuropsychological and neuroimaging literatures. On the one hand, functional activations do not prove the necessity of a region . On the A meta-analysis of 8 cognitive domains found a tripartite domain-general organization of parietal function. Five principles of parietal organization were revealed, which cannot be captured in their entirety within existing theoretical accounts of parietal function. A new framework is proposed , which assimilates many existing notions and combines them with computational and connectivity insights. Specifically, out of domain-general multimodal buffering, a tripartite parietal division emerges as a consequence of differential long-range connectivity. Furthermore, long-term statistical extraction from repeated buffering generates the various verbal and nonverbal representations that are associated with the parietal lobe.Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.Medical Research Council (UK) grant to MALR (MR/J004146/1). Funding to pay the Open Access publication charges for this article was provided by a UK Research Councils (RCUK) award to the University of Manchester.This study was supported by a


Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. They are involved in early steps of lymphocyte activation through their SH2-mediated transient interaction with signal transducing effector molecules, such as Cbl, ZAP-70, CasL, and STAT5. In addition, they constitutively associate, via their SH3 domain, with effector molecules, such as C3G, that mediate cell adhesion and regulate lymphocyte extravasation and recruitment to sites of inflammation. Recent studies demonstrated that the conformation and function of CrkII is subjected to a regulation by immunophilins, which also affect CrkII-dependent T-cell adhesion to fibronectin and migration toward chemokines. This article addresses mechanisms that regulate CrkII conformation and function, in general, and emphasizes the role of Crk proteins in receptor-coupled signaling pathways that control T-lymphocyte adhesion and migration to inflammatory sites. Adaptor proteins are essential components of signal transduction pathways in all cell types due to their ability to link engaged receptors to specific downstream signaling cascades. The presence of multiple protein–protein interaction domains in an adaptor protein enables the simultaneous interaction with two or more effector molecules and the spatially and temporally orchestrated assembly of multimolecular complexes that are essential for signal transduction.The mammalian CT10 (chicken tumor virus number 10) regulator of kinase (Crk) adaptor proteins, CrkI and CrkII, are alternatively spliced products of a single gene –4. CrkIIThe Crk proteins are ubiquitously expressed and act as major convergence points of tyrosine kinase signaling pathways. They utilize their SH2 and SH3 domains for interaction with multiple effector molecules and integrate molecular information obtained from diverse sources, such as growth factors, extracellular matrix (ECM), pathogens, and apoptotic cells . In geneCrk proteins possess two types of basic modules that function as protein–protein interaction domains. The Crk amino-terminus SH2 domain binds tyrosine-phosphorylated proteins containing the preferred consensus motif, pY-x-x-P, whereby specificity is provided by the two to five residues C-terminal of the phosphotyrosyl group  Figure . In accoA second module found in all Crk proteins is an SH3 domain, which binds polyproline-rich sequences, with a preference toward the P-x-L-P-x-K motif  Figure . In T ceThe SH3C of both CrkII and CrkL possesses a consensus SH3 sequence homology, but its binding surface contains some hydrophobic residues that substantially reduce its affinity to polyproline-rich sequences . Two indMore recent studies suggested that the main function of the SH3C domain is directed at stabilizing CrkII and/or CrkL in a conformation that negatively regulates their biological function –29.Apart from the SH2 and SH3 domains, Crk proteins contain additional regulatory motifs that may have an impact on the protein conformation and biological activity Figure . One sucThe conserved tyrosine residues in CrkII and CrkL reside within a Y-x-x-P sequence, and upon phosphorylation, they internally interact with the SH2 domain, thus operating as dominant-negative binding motifs . NMR spein vitro interaction of the full-length non-phosphorylated CrkII with Abl SH3 domain was rather weak, phosphorylation of CrkII-Tyr221, or addition of a phosphopeptide corresponding to the CrkII-Tyr221 phosphorylation site, stimulated CrkII association with the Abl SH3 domain. The results suggested that the proline-rich insert within the Crk SH2 domain constitutes an SH3 domain-binding motif which is regulated by phosphopeptide ligand binding to the Crk SH2 domain. CrkI possesses an identical SH3N domain allowing it to associate with Abl, but due to the lack of the linker region that includes Tyr221 (in addition to the entire SH3C domain), it cannot undergo phosphorylation in vivo, and apparently exhibits a very low affinity to the Abl SH3 domain.A second regulatory motif within an extended loop in the SH2 domain possesses a proline-rich region, which is dispensable for phosphopeptide binding and is able to interact with various SH3 domains, including that of the Abl PTK , 35, 36.cis–trans isomerases (PPIases) . Acquisition of a cis conformation resulted in an intramolecular interaction between the two SH3 domains of CrkII, forming a closed conformation of an autoinhibited molecule. In contrast, the CrkII trans conformer acquired a relatively open and extended conformation that was readily available for interaction with SH2- and SH3N-binding partners. PPIases catalyze both cis-to-trans and trans-to-cis interconversion of CrkII, thereby determining the temporal and spatial activation of CrkII and CrkII-regulated signaling events.Recent studies revealed an additional regulatory motif within the linker region of the chicken, but not human, CrkII, which serves as a target for peptidyl-prolyl PPIases) , 37, 38.In vitro coincubation of CypA with human T-cell-derived CrkII increased CrkII binding to C3G while immunophilin inhibitors [such as cyclosporine A (CsA) and FK506] downregulated CrkII binding to C3G, suggesting that CrkII-C3G interaction may be subjected to a regulation by immunophilins (1-236 sandwiched between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The basal FRET level of these cells was further augmented by CsA and FK506 treatment, reflecting a drug-induced inhibition of immunophilins and a trans-to-cis conversion of CrkII.More recent studies demonstrated that human T-cell-derived CrkII, but not CrkI, can physically interact with representatives of two families of PPIases, CypA and FK506 binding protein (FKBP), collectively termed immunophilins . In vitrophilins . This asThe differential sensitivity of the truncated chicken vs. human CrkII to PPIases was attributed to the nature of the amino acid that immediately follows the Pro238 (chicken)/237 (human) residue, Phe and Ile in the chicken and the human CrkII, respectively , 29, 38.CrkII shares with CrkL a high degree of sequence homology and ligand-binding preference, but the two proteins have distinct physiological roles. Structural studies demonstrated that the two proteins exhibit a distinct intramolecular assembly of their domains , 40, whiAnother line of studies demonstrated that the extent of expression of Crk is subjected to a regulation by microRNA (miRNA) . These sl-selectins, CD44, and CD31  and adhere to vascular endothelial cells is absolutely essential for the induction of all types of T-cell-mediated immune responses. Accordingly, deficiency in adhesion molecules and/or their signaling pathways, such as in leukocyte adhesion deficiency (LAD), results in impaired or complete absence of immune responses . InteracA large number of T-cell effector molecules play a role in the transduction of signals regulating the activity of adhesion receptors , 45–47. The CasL docking protein  is linkeCrk and CrkL gene conditional knockout mice , are integral components of the signaling pathway that promotes Rap1 activation and chemokine-induced T-cell migration , 64. AblThe Chat-H is a member of the novel SH2-containing protein (NSP) family , also knin vitro invasion of the hybridoma cells, inhibited their ability to disseminate in vivo and form remote metastases, and suppressed their ability to migrate toward the chemokine, stromal cell-derived factor 1 (SDF-1)  . ZAP-70  (SDF-1) , 70, 72  (SDF-1) , 12, 73, (SDF-1)  and TCR cis–trans isomerization controlled by immunophilins  adhesion receptors (An additional effector molecule that regulates T-cell adhesion by alteration of Crk-dependent signaling pathways is the E3-ligase, Cbl . The Cbleceptors  as well eceptors . Taken teceptors .The Crk adaptor proteins play an essential role in the regulation of T-cell adhesion and migration to sites of inflammation. Together with C3G and CasL, they coordinate signaling pathways leading to activation of Rap1 that upregulate the clustering and affinity of integrins to their ligands. This general mechanism might be relevant to the regulation of some, but not all, integrins since different T-cell integrins might be coupled to distinct effector proteins. The mechanism of regulation of the stoichiometric ratio between CrkI, CrkII, and CrkL in specific cell types is unknown, and the contribution of individual Crk isoforms to the adhesion response has not been thoroughly studied. Further investigations are also required in order to determine the potential regulation of selected Crk proteins by isomerases and whether inhibitors of immunophilins can downregulate T-cell adhesion and migration irrespective of their inhibition of the calcineurin/NFAT pathway.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.


For over a decade, controlling domain-wall injection, motion and annihilation along nanowires has been the preserve of the nanomagnetics research community. Revolutionary technologies have resulted, like racetrack memory and domain-wall logic. Until recently, equivalent research in analogous ferroic materials did not seem important. However, with the discovery of sheet conduction, the control of domain walls in ferroelectrics has become vital for the future of what has been termed ‘domain-wall electronics'. Here we report the creation of a ferroelectric domain-wall diode, which allows a single direction of motion for all domain walls, irrespective of their polarity, under a series of alternating electric field pulses. The diode's sawtooth morphology is central to its function. Domain walls can move readily in the direction in which thickness increases gradually, but are prevented from moving in the other direction by the sudden thickness increase at the sawtooth edge. In ferromagnets, domain wall control is vital for applications like racetrack memory, whereas in ferroelectrics it could facilitate new electronics. Here, the authors demonstrate a diode for ferroelectric domain wall motion, where domain walls can move in one direction but not in the other. This is not trivial. If a conventional field were applied to a set of domains under normal circumstances, those with dipoles oriented favourably with respect to the field would grow, while others would contract, such that domain walls of opposite polarity (defined by the specific sequence of adjacent domain orientations) would move in opposite directions . In Cowb2122et al.An alternative shift register concept has been suggested and demonstrated by Franken et al.26Allwood 4 (KTP) and integrated into a coplanar capacitor geometry  was manufactured from a bulk single crystal of uniaxial ferroelectric KTiOPOgeometry . The tergeometry . After cThe ferroelectric diode structure was therefore designed with a relatively thin parallel-sided region, to encourage domain-wall injection, adjacent to a section of gradual thickness increase up to a maximum point, followed by a sudden drop to another parallel-sided region of intermediate thickness ; a schematic is shown in 29et al.It should be noted that throughout the study described, it has been assumed that the domain walls examined are all flat planar objects and that their position mapped at the top surface of the KTP is representative of that through the entire thickness of the ferroelectric; while this assumed planar geometry should minimize the domain wall area and hence energy, which has been shown to be central to the successful operation of the wedge diode, some positional meandering through thickness could, in principle, occur. Indeed, finite element modelling shows that minor local variations in field distribution are present as a function of thickness, so that domain walls at the bottom surface of the KTP experience a slightly different environment from those at the top. In particular, in the thicker regions of the wedge structure, small hot spots at the electrode–ferroelectric interface begin to develop, which are not obvious in thinner regions. These hot spots might be thought to be likely points for domain-wall injection as demonstrated in previous researchet al.In summary, the notion that the total energy of a domain wall is proportional to its area has been used to design a sawtooth energy landscape through machining a ferroelectric lamella with a sawtooth thickness profile. This lamella has been integrated into a capacitor structure and the domain dynamics resulting from pulsed electric fields monitored using PFM. Distinct asymmetry in the domain-wall mobility has been demonstrated: domain walls can progress readily up the shallow incline of the sawtooth, but are prevented from moving in the opposite direction by the steep potential gradient at the sawtooth ledge. Effectively, a domain-wall diode has been demonstrated; by extension, in principle, the possibility of a shift register structure for ferroelectrics, analogous to those critical for domain-wall logic and racetrack memory applications in nanomagnetics, has also been shown. The fabrication methodology presented is clearly only relevant for a proof-of-principle device. However, the fact that Franken 2O3 substrates. Thermal annealing (300 °C for 6 h) was used to partially recrystallize the FIB-induced damage at the surface of the lamellae and expel gallium in the form of oxide platelets, which was removed via 3 min of acid etching in a 3 M HCl solution. Localized electron beam deposition of platinum at lamella edges  ensured good electrical contact and mechanical robustness of the completed coplanar capacitor device. Any deliberate local variations in lamellar thickness were developed on the top surface only, as a flat lower surface was needed to ensure sound electrical contact. Finally, each electrode of the sample was conductively secured with silver paste onto a glass slide with macroscopically patterned sputter-coated platinum electrodes, enabling application of voltage across the sample, whilst under an AFM scanner head.All capacitor structures investigated were fabricated by cutting lamellae from a bulk single crystal of KTP, using a FEI200TEM FIB, and placing them across a FIB-milled interelectrode gap (2 μm in width) on platinum-coated AlRMS at a frequency of 20 kHz was applied to a Nanosensors PPP-EFM cantilever (force constant ∼2.8 N m−1). The d.c. voltages were applied to lamellae using a Keithley 237 High Voltage Source Measure Unit.PFM measurements were carried out using a Veeco Dimension 3100 AFM system with a Nanoscope IIIa controller, modified for PFM measurements using a EG&G 7265 lock-in-amplifier. Here an oscillating voltage of 2 VHow to cite this article: Whyte, J. R. & Gregg, J. M. A diode for ferroelectric domain-wall motion. Nat. Commun. 6:7361 doi: 10.1038/ncomms8361 (2015).


Keap1 is a highly redox-sensitive member of the BTB-Kelch family that assembles with the Cul3 protein to form a Cullin–RING E3 ligase complex for the degradation of Nrf2. Oxidative stress disables Keap1, allowing Nrf2 protein levels to accumulate for the transactivation of critical stress response genes. Consequently, the Keap1–Nrf2 system is extensively pursued for the development of protein–protein interaction inhibitors that will stabilize Nrf2 for therapeutic effect in conditions of neurodegeneration, inflammation, and cancer. Here we review current progress toward the structure determination of Keap1 and its protein complexes with Cul3, Nrf2 substrate, and small-molecule antagonists. Together the available structures establish a rational three-dimensional model to explain the two-site binding of Nrf2 as well as its efficient ubiquitination.  •Keap1 and Nrf2 are multidomain proteins that determine the antioxidant response.•Structural models suggest how Keap1 assembles into a Cullin–RING ligase.•Dimeric Keap1 supports a two-site tethering mechanism for Nrf2 ubiquitination.•Crystal structures reveal the mechanism of action of chemical inhibitors of Keap1. In addition, it controls transporters critical for cellular detoxification. Consequently, the Keap1–Nrf2 system has emerged as an important therapeutic target in cancer and neurodegenerative conditions, as well as many autoimmune and inflammatory diseases The Nrf2 protein in humans is 605 amino acids long and contains seven highly conserved regions known as Nrf2-ECH homology (Neh) domains . Neh1 coDrosophila proteins Broad complex, Tramtrack, and Bric à brac, in which it was first identified Keap1 belongs to the BTB-Kelch family of proteins, which comprises some 50 members variously named as Kelch-like 1–42 (KLHL1–42) or Kelch and BTB domain-containing 1–14 (KBTBD1–14) To date there are no available high-resolution structures describing either of the full-length Keap1 or Nrf2 proteins. Nonetheless, a number of crystal structures featuring Keap1, or its BTB-Kelch family homologs, have revealed the molecular mechanisms determining its interactions with Nrf2 substrate or Cul3 protein, as well as the action of chemical inhibitors that stabilize Nrf2 for therapeutic gain.2KD value ~5 nM) ETGEFL) incorporating amino acids 69–84 of Nrf2, which flank the conserved ETGE motif Nuclear magnetic resonance spectroscopy has shown the Neh2 region of Nrf2 to be intrinsically disordered Overall, the Kelch domain contains six Kelch repeats that fold into a six-bladed β-propeller structure 2  as well as β-strands B and C (BC loop). The bound ETGE peptide of Nrf2 adopts a β-turn conformation that inserts into this pocket to establish a buried surface area of 420 Å2 A [36], [10 helices (Ile28 to Leu30 and Arg34 to Phe37). In this revised structure, the DLG motif residues Asp29–Leu30–Gly31 are located similar to the ETGE motif residues Glu79–Thr80–Gly81.The conserved DLG motif in the Neh2 region of Nrf2 was identified as a second independent binding site with 100-fold weaker affinity for Keap1 Comparison of the DLG and ETGE peptide complexes shows that they insert in a manner similar to the bottom of the Kelch domain pocket, but that they deviate at their N-terminus owing to the helical conformation of the DLG peptide motif C. ThermoThese differences are thought to be important for fine-tuning regulation of the stress response The Kelch domain structures also help to explain the effects of somatic cancer mutations identified in Keap1 A and 2B,3The BTB domain is a protein–protein interaction domain found throughout eukaryotes as well as in the poxvirus zinc finger (POZ) protein family and is consequently sometimes referred to as the BTB/POZ domain 2. Notable among these are the long α1 helix and the N-terminal β1 strand, which forms a domain-swapped antiparallel β-sheet with the β5 strand of the other bound subunit. Some members of the BTB-Kelch family have been reported to heterodimerize, such as the KLHL9 and KLHL13 protein pair The structure of the BTB domain of Keap1 was reported in 2014 and shows a fold similar to the PLZF domain despite its low sequence identity and divergent cellular function 4The E3 ligase activity of Keap1 requires its assembly with Cul3 to become part of a larger CRL3 complex The Cul3 N-terminal domain contains three Cullin repeats, each folded as a five-helix bundle, as well as an N-terminal extension of some 24 amino acids The 3-box motif connects the BTB domain to the central portion of the Keap1 protein known either as the IVR domain or the BACK  domain 5A model of the full-length Keap1 homodimer has been obtained at low resolution by single-particle electron microscopy A more complete model of the full CRL3 complex  has been6Chemical inducers of Nrf2 that block Keap1 function have proven an effective mechanism to exploit the antioxidant response in the fight against human disease. Indeed, the Nrf2 inducer BG-12, a formulation including dimethyl fumarate, was recently approved for the treatment of relapsed multiple sclerosis Other electrophiles are expected to bind to Cys273 and Cys288 in the IVR domain More recently, a number of cocrystal structures have been reported for small-molecule inhibitors that target the Kelch domain noncovalently and compete directly for binding with the Nrf2 peptide 7A combination of biochemical and structural studies has yielded remarkable insights into the molecular mechanisms controlling the cellular response to oxidative and electrophilic stress. Importantly, the structural models are consistent with the proposed two-site hinge and latch model for Nrf2 capture and reveal sensible spatial arrangements for its subsequent ubiquitination. Their construction is particularly enabled by the modular nature of the Cullin–RING ligases, which allows different structures to be assembled together for an understanding of the entire complex. However, the lack of a high-resolution full-length Keap1 structure currently limits our knowledge in several respects. Progress here is perhaps hindered by the short linker that connects the IVR and Kelch domains. For example, the structure of the MATH-BTB protein SPOP shows an analogous linker that is highly flexible leading to asymmetry in the dimeric protein, as well as the E3-substrate complex Overall, the lessons learned from the Keap1–Nrf2 system provide a prototype model for our understanding of the entire BTB-Kelch family. Structural studies across this family are less advanced, but already reveal a diversity in the Kelch domain substrate pocket, as well as alternative peptide binding conformations and interfaces