Plasmodium falciparum virulence has been ascribed to its ability to sequester in deep vascular beds, mediated by the variant surface antigen family PfEMP1 binding endothelial receptors like ICAM-1. We previously observed that naturally-acquired antibodies that block a PfEMP1 domain, DBL2β of PF11_0521 allele, from binding to the human ICAM1 receptor, reduce the risk of malaria hospitalization in children. Here, we find that DBL2βPF11_0521 binds ICAM-1 in the low nM range and relate the structure of this domain with its function and immunogenicity. We demonstrate that the interaction with ICAM-1 is not impaired by point mutations in the N-terminal subdomain or in the flexible Loop 4 of DBL2βPF11_0521, although both substructures were previously implicated in binding ICAM-1. These data will help to refine the existing model of DBLβ::ICAM-1 interactions. Antibodies raised against full-length DBL2βPF11_0521, but not truncated forms lacking the N terminal fragment, block its interaction with ICAM-1. Our data suggest that full length domain is optimal for displaying functional epitopes and has a broad surface of interaction with ICAM-1 that is not disrupted by individual amino acid substitutions at putative key residues. This information might be important for the future design of anti-malarial vaccines based on PfEMP1 antigens. The sequestered mass of adherent parasites leads to vascular occlusion that strongly correlates with disease severity Cytoadherence plays an important role in the lifecycle and virulence Each PfEMP1 protein consists of single intracellular and trans-membrane domains, and several extracellular domains  heavily cross-linked by disulfide bonds. All PfEMP1 domains have been classified into several sub-classes: Duffy binding like (DBL) α, β , γ, δ, ζ, ε, and X; and cysteine-rich interdomain regions (CIDR) α, β, and γ Various refinements of domain classification and of domain boundaries resulted from recent analyses of PfEMP1 sequences from 7 parasite genomes in vitroPF11_0521 predict reduced risk of hospitalization for malaria among Tanzanian children Though its role remains inconclusive, ICAM-1 has been proposed as the key host adhesion receptor for iRBC causing CM in silico, based on several lines of evidence, such as the key role of a single amino acid residue in loop 4 of the DBL2β domain in silico model) is important for functional activity PF11_0521 domain to assess their roles in binding ICAM-1. We also examined the functional anti-adhesion activity of antibodies raised against truncated and full-length variants of DBL2βPF11_0521 to better understand the requirements for PfEMP1 domain-based vaccines that might prevent iRBC adhesion and thus severe malaria.Interactions between ICAM-1 and the DBL2β domain were previously modeled Guide for the Care and Use of Laboratory Animals  and applicable Seattle Biomedical Research Institute (Seattle BioMed) policies and procedures. Seattle BioMed has an Animal Welfare Assurance (A36640-01) on file with the NIH Office of Laboratory Animal Welfare. Protocols #AO-06-ABP and #AO-02 have been approved by Seattle BioMed IACUC committee. All animals used in the experiments were observed on a daily routine for presence or absence of distress and/or signs of illness, timely veterinary care was provided as needed. Euthanasia method via exsanguination by cardiac puncture under ketamine/xylazine anesthesia was used.Animal ethics adhered to specific national and international guidelines. Animal use protocols “Antibodies Inc. Protocol (Offsite)” and “Liver Stage Vaccines” meet the standards of the PF11_0521 domain and its expression in COS-7 cells as a surface-expressed molecule was described in PF11_0521 domain expressed in COS-7 cells and several domain structural features, as well as constructs (expressed in E. coli) used in this work. The nucleotide and corresponding protein sequences for this domain, the various truncation and point-mutation constructs, as well as names of relevant cloning vectors are all shown in Cloning of the full-length DBL2βE. coli, purified by Ni-column followed by Reverse Phase HPLC and refolded as described earlier Full-length DBL2β domain  and its truncated fragments AA 91–391 (“short”), AA 267–391 (“3-helix”), and the N-terminal sub-domain  were cloned into the pET28b vector, expressed in E. coli using pET28b with subsequent purification and re-folding by the same methods described above for full-length DBL2βPF11_0521 domain.We expressed HisAdEx E.coli-expressed/refolded domain (see below), and this amount then used to normalize ICAM-1-binding levels E. coli-expressed and purified domains were immobilized on carboxy-modified polystyrene beads (BioRad) using the manufacturer-supplied protocol and assayed in the same fashion as bead-immobilized COS-7-expressed proteins. COS7-expressed GFP-fused proteins were immobilized on the beads through capture by anti-GFP antibodies previously immobilized by chemical cross-linking E. coli and COS-7 expressed proteins, the amount of protein bound to the beads was measured by reactivity with anti-full length PF11_0521DBL2β domain sera (rat sera to E.coli-expressed domain) as described below.We performed binding and binding-inhibition studies as previously described, using BioPlex (BioRad) approach, COS-7 expressed proteins, and ICAM-1-Fc chimera (R&D Systems) E. coli-expressed domain variants were used for preparation of antibodies in rats at Antibodies, Inc.  using their standard schedule protocol and 10 µg of antigen per injection, and in mice at Seattle BioMed as described earlier Ethical Statement above and has been approved by IACUC at Seattle BioMed.Purified and refolded A range of dilutions of anti-sera raised against various domain constructs was incubated with different bead-immobilized domain constructs for 1 hour at room temperature (RT). Pooled pre-immune sera and anti-sera from animals raised against control proteins (HisAdEx or AMA1) were used as negative controls. We used pooled sera from all animals immunized with the same antigen. Beads were washed 3 times with phosphate buffered saline (PBS)-0.05% Tween-20 (PBST), incubated with goat anti-species IgG coupled to PE  for 1 hour at RT, washed 3 times in PBST and 1 time with PBS, and finally re-suspended in 125 µl of PBS. Signals were measured on BioPlex 200 (BioRad) as described in our publications E. coli) and immobilized on BioPlex beads (as described above) for these measurements. E. coli-expressed protein was refolded as described above. Initial velocity of binding was measured at various concentrations  of ICAM-1-Fc. At these concentrations and incubation times (5 to 35 min), the level of ICAM-1 binding to bead-immobilized domain was less than 10% of the maximum. The incubations and measurements were performed in a homogenous system; ICAM-1-Fc was pre-incubated for 1 hour with an excess of donkey anti-human IgG coupled to phycoerythrin (PE) detection molecules, and then added to beads with immobilized full-length DBL2β domain. Final concentrations of ICAM-1 in these mixtures are shown above and the final dilution of anti-human IgG-PE was 1∶250. After incubation for the indicated periods of time, beads were subjected to signal measurements using a Bio-Plex 200 machine (BioRad) without any washing steps. The actual time (in seconds) between mixing ICAM-1-Fc and donkey anti-human IgG-PE with beads and signal measurement events on the machine was used for kinetics calculations. As active ICAM-1-Fc molecules are organized as dimers, we used its molecular mass equal to ∼200 kDa (R&D Systems) for calculations. We used the Michaelis-Menten equation and Lineweaver-Burk plots D. Linear regression lines and 95% confidence intervals (CI) were calculated using GraphPad Prizm software.We used full-length DBL2β domain expressed in both systems .DBL2β::ICAM-1 interactions suggested by Bertonati and Tramontano A model of However, the Bertonati-Tramontano model has limitations, and several observations cannot be explained by this model. For example, the Y-motif in the C2 part of the domain  was showin silico substitutions of amino acid residue 286 in the model in vitro the effect of substitutions of amino acid residue 286 on DBL2β::ICAM-1 interactions.Different parasite strains P. falciparum genomes PF11_0521 domain, which strongly and specifically binds ICAM-1, we reported that the N-terminal subdomain contributes to ICAM-1 binding activity Finally, we have shown in silico modeling of Tyr substitution (above) that predicts an additional intra-molecular hydrogen bond (between Tyr 186 and Arg113 of the domain) that may stabilize the complex  these: the full length domain , most likely due to incorrect folding of these truncated domain fragments (see below). Refolded full-length recombinant domain strongly bound the ICAM-1 receptor  was ∼3–fold lower than that seen with COS-7-expressed protein. This indicates that only a fraction of the E. coli preparation (∼33%) is refolded correctly to the active conformation or is sufficiently stable for binding on the beads. Random immobilization (of the E. coli-expressed protein) may inactivate some number of molecules by their unfavorable cross-linking on the surface of the beads. This may contribute to the inactive pool of bead-immobilized domain molecules and indicates that at least 33% of the refolded soluble preparation was active before cross-linking. Directed immobilization (of the COS-expressed domain) in this respect is preferable over the random immobilization.In accordance with our previous results using COS-expressed truncated domains receptor , though PF11_0521 domain and ICAM-1 receptor is strong, with a calculated equilibrium dissociation constant (KD) in the low nM range  (Binding between the DBL2β–4.4 nM) . The higPF11_0521 domain are common in malaria-immune adults PF11_0521 domain would affect binding of this domain to ICAM-1, and as well to determine the structure and size of the domain needed to re-create functional epitopes. All four E. coli-expressed DBL2βPF11_0521 domain constructs  were used for immunizations of rodents, as was plasmid DNA pHisAdEx-DBL2βPF11_0521 containing full length domain. This latter construct was previously used for production of functionally active protein in COS-7 cells We have previously demonstrated that antibodies against DBL2βE. coli and COS-7 expressed full-length domains . Antisera collected after DNA immunization failed to react to the “short” E. coli expressed truncated domain, which represents a substantial portion of the full-length protein but, as we suggested, is mis- or un-folded. We conclude that intramuscular DNA immunization yielded correctly folded domain on the surface of the host cells and induced antibodies that predominantly recognize surface-exposed structural epitopes of this correctly folded form, whereas antibodies raised against E. coli-expressed protein (see below) recognize epitopes of both folded and unfolded protein and might therefore reflect total protein in a preparation rather than solely the binding form of protein. Because antibodies raised by DNA immunization recognize only the folded form of the domain, they cannot be used to determine the amount of total protein immobilized on the beads.Mouse anti-full-length domain antibodies prepared by DNA immunization recognized both  domains , with siE. coli expressed/refolded full-length domain recognized both E. coli expressed/refolded full length domain and COS-7 expressed domain  binding activity of mutant variants, and compared specific ICAM-1 binding activity of E. coli expressed/refolded and COS-7 expressed protein (above).Rat antisera against d domain , with tid domain , presumaE. coli expressed domains but recognition of COS-7 expressed full-length domain was significantly weaker  (E. coli-expressed NTF and full-length domain but poorly recognized COS-expressed full-length domain and did not inhibit ICAM-1 binding (data not shown).Rat anti-“short” antibodies strongly and comparably recognized full-length and “short” n beads) , top, agn beads) . Similarn beads)  but had n beads) , bottom.E. coli expressed protein) were functionally active as they can completely inhibit ICAM-1 binding to COS-expressed DBL2β domain Click here for additional data file.Figure S2Structural elements of interacting ICAM-1 and DBLβ in the model of Bertonati and Tramontano. Model A) and amino acid substitution in loop 4 (B) was modeled using Deep View Swiss-PDB viewer. In panel A loops 1 through 4 participating in interactions of DBLβ domain with ICAM-1 are shown in red, the rest of the molecule in green, ICAM-1 molecule in yellow. Ala286 residue in loop 4 that is involved in contacts with ICAM-1 is shown in blue. Y motif is circled. In panel B amino acid residue 286 (Ala or Tyr) is shown in pink. Amino acid residues Asn (N) and Tyr (Y), which are located at the beginning and the end of the loop 4, are indicated. Residues in the vicinity of the position 286 are shown in space-filling shape with CPK coloring. Substitution Ala286Tyr creates an additional hydrogen bond (arrowhead in panel C) between Tyr286 and Arg113 (numbers according to (PPTX)Click here for additional data file.Figure S3PF11_0521.Predicted secondary structure of N-terminal sub-domain and amino acid residue mutations in the first α-helix of DBL2β ICAM-1 binding and non-binding domains (PPTX)Click here for additional data file.Figure S4D for ICAM-1 binding to PF11_0521 DBL2 domain.Determination of equilibrium dissociation constant K Kinetics of binding for both E. coli expressed/refolded  and COS-7 expressed  domains was measured in 4 independent experiments. 95% confidence intervals (CI) shown by dashed lines. Error bars are standard errors of mean. N, number of replicates for each concentration point. Formulas show linear regressions. V, initial velocity of binding; C, concentration of ICAM-1; min, minutes, AU, arbitrary units.(PPTX)Click here for additional data file.Table S1PF11_0521 and antibodies against these constructs.Summary of results of binding and inhibition of binding of ICAM-1 receptor obtained with various constructs of DBLβ(DOCX)Click here for additional data file.


Single nucleotide polymorphisms (SNPs) are a major contributor to genetic and phenotypic variation within populations. Non-synonymous SNPs (nsSNPs) modify the sequence of proteins and can affect their folding or binding properties. Experimental analysis of all nsSNPs is currently unfeasible and therefore computational predictions of the molecular effect of nsSNPs are helpful to guide experimental investigations. While some nsSNPs can be accurately characterized, for instance if they fall into strongly conserved or well annotated regions, the molecular consequences of many others are more challenging to predict. In particular, nsSNPs affecting less structured, and often less conserved regions, are difficult to characterize. Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs. Binding sites targeted by the PDZ modular peptide recognition domain have recently been characterized. Here we use this data to show that it is possible to computationally identify nsSNPs in PDZ binding motifs that modify or prevent binding to the proteins containing the motifs. We confirm these predictions by experimentally validating a selected subset with ELISA. Our work also highlights the importance of better characterizing linear motifs in proteins as many of these can be affected by genetic variations. Genome sequencing projects have uncovered thousands of genetic variations in human populations 6. It is therefore important to understand and predict the functional and molecular consequences of nsSNPs and several computational approaches have been developed to address this issue SNPs can be rapidly identified using microarray or DNA sequencing technology and the number of reported nsSNPs in human proteins exceeds 10Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs Linear motifs are not easy to detect in a statistically significant way because they are short and remain poorly annotated in the human genome. One standard approach to identify them is to use consensus motifs  and large collections of such motifs are available in databases Recently, together with an increase in available experimental data for these domains e.g. , more elHere we show that these models of specificity are also useful to determine nsSNPs affecting PDZ-binding linear motifs in human proteins. We observed several cases of nsSNPs that fall into PDZ-binding motifs and where the mutated residue is expected to significantly disrupt the interaction. Experimental validations for three of the most promising cases were carried out with enzyme-linked immunosorbent assays (ELISAs) to assess binding activity. In all three cases, the WT peptides were found to interact with the predicted PDZ domain, while the nsSNPs significantly disrupted the interaction. In particular, we found that the PDZ domain of CYTIP  can bind the C-terminal peptide of PCDHA1, a member of the cadherin family. The PDZ domain of CYTIP is required for its correct localization to the cell cortex All nsSNPs from dbSNP To scan human proteins for the presence of a PDZ binding motif, we first retrieved all human proteins from RefSeq . As PDZ domains occur primarily in cytoplasm, we filtered out extracellular and mitochondrial proteins using Gene Ontology categories In total our filtering procedure resulted in 19,751 proteins with 13,490 nsSNPs consisting of a single residue variation in a C-terminus and 26,864 stop codons resulting in a different C-terminus. When computing statistics regarding nsSNPs in C-terminal regions, the frequency of nsSNPs in C-termini was compared to the frequency of nsSNPs anywhere in proteins using the Binomial test, since multiple nsSNPs can affect the same residue.Positions Weight Matrices (PWMs) were used to predict PDZ-binding motifs containing C-termini. For each PDZ domain, PWMs were built from phage display data i and a protein j is mathematically given by: j(n) is the amino acid at position n in the C-terminus of peptide j, and n and amino acid j(n) for the ith PDZ domain. The higher the score, the more similar to the PDZ binding specificity the C-terminal sequence is. Hence, for a given threshold on PWM scores, C-terminal regions are predicted to contain a PDZ-binding motif if the PWM score is higher than the threshold value for at least one PDZ domain (maxS) C-terminal sequences and where the mutated sequence leads to a much lower score  are significantly underrepresented .The aim of our work is to develop a computational method to identify nsSNPs most likely to disrupt or create PDZ-binding motifs. As there are currently no large experimental dataset of binding activity values between WT and naturally mutated PDZ ligands to train our model, some of the thresholds, in particular base see  [36]. Inbase see . 23 out 3PO4 and the signal recorded at 450 nm in a Microplate reader .Biotinylated peptides were synthesized consisting of either the wild-type C-terminal sequence or the nsSNPs predicted to disrupt the interactions with PDZ domains. Assay plates were prepared by immobilizing the biotinylated peptide 50 μg/ml in phosphate buffered saline (PBS) on Maxisorp immunoplates coated with neutravidin  and blocked with bovine serum albumin (0.5% w/v BSA in PBS). After 1 h incubation with GST-PDZ fusion protein at the indicated concentrations see , the pla−26, Binomial test), likely because protein C-termini are generally solvent-accessible and are less constrained in protein structures −21).nsSNPs replacing one amino acid by another one can have different effects on proteins depending on which part of the amino acid sequence they affect. For instance, nsSNPs found in protein interaction interfaces will often result in weakening or disruption of the interaction. To gain insight into nsSNPs affecting PDZ-mediated interactions, we first computed the frequency of nsSNPs in C-terminal regions, as these are the classical targets of PDZ domains. C-terminal sequences are defined here as the last seven residues of proteins, following Tonikian et al. We then used the PWMs derived from phage display data to scan wild-type human C-termini  and rank them according to their score with each PDZ domain (see Methods). High scoring C-termini contain well-defined PDZ binding motifs and are predicted to interact with some PDZ domain minS, maxS] on the PWM scores ijS (see Method), we classified nsSNPs in three categories: predicted to disrupt PDZ-binding, predicted to create a new PDZ-binding motif and predicted not to affect possible PDZ-mediated interactions (minS) from which C-terminal sequences are likely not to bind to a PDZ domain, we used the results of minS = −8.3. This corresponds approximately to the point where nsSNPs start being no longer underrepresented in PDZ-motif containing C-terminal sequences. Moreover, reported interactions between PDZ domains and WT C-termini scoring lower than minS are significantly underrepresented .To gain insight into the effect of these mutations, we compared the similarity of both the wild type and mutated C-termini to the optimal PDZ domain specificity modeled by the different PWMs. By defining a confidence interval [ractions . To fix maxS defining PDZ-binding motifs . In total, we identified 41 cases of PDZ motif-containing C-terminal sequence that were significantly disrupted by nsSNPs. In addition, we found 20 cases of nsSNPs falling in C-termini not predicted to bind PDZ domains, but for which the mutated sequence shows a clear PDZ-binding motif  or create (279 STOP codons) a C-terminal sequence predicted to bind some PDZ domain  Using the same threshold parameters main see . Many ofPredicting the functional consequences of genetic variations is important for understanding the molecular mechanisms underlying both hereditary diseases and differences between populations. For SNPs outside of coding regions or SNPs not affecting the encoded amino acids, interpretation of functional effects is challenging, though changes in promoter or enhancer regions, DNA structure, or gene expression can sometimes explain their functional effects. Large-scale initiatives such as the ENCODE project are playing an important role to decipher these molecular mechanisms Using a detailed computational model of PDZ domain binding specificity, we have shown that the effect of nsSNPs affecting PDZ-binding linear motifs could be accurately predicted. This problem is particularly important first because PDZ domains mediate key protein-protein interactions and second because many nsSNPs affect C-terminal sequences and some of them have already been linked to diseases Our experimental validation shows that accurate predictions can be achieved for nsSNPs found in linear motifs. The experimentally observed interactions also provide interesting evidence of potentially yet undetected protein interactions. Most promising for follow-up experiments is the possible interaction between the CYTIP PDZ domain and the C-terminal peptide of PCDHA1 .Throughout this work, we have considered the last seven C-terminal amino acids as the maximum length of PDZ-binding motifs. This encompasses specificity-determining positions for most PDZ domains maxS. However, in these cases, the effect of nsSNPs is more difficult to evaluate since changes at the C-terminal sequence do not result in large differences in PWM scores. Moreover, some PDZ domains display specificity profiles that do not match existing C-termini  Our predictions, especially the examples listed in The presence of nsSNPs in a protein can be interpreted in multiple ways. First, for nsSNPs with high population frequency , the different variants may not have a major deleterious functional impact and may be well tolerated. Alternatively, nsSNPs linked to diseases or other phenotypes often have some molecular consequence such as modifying enzyme activity, destabilizing protein structures or disrupting protein interactions In this work we have focused on PDZ domains, mostly because of the availability of experimental data that enable accurate computational predictions of interactions. Yet, the strategy presented in this work could be applied to other domains such as SH3, WW or kinases. With the current increase in available genomic data coming for large population sequencing projects and cancer genomics projects, genetic variations will increasingly be uncovered. Methods such as ours, eventually expanded to consider a wider range of protein interaction domains, will help decipher the molecular mechanisms resulting from these new nsSNPs.Table S1List of PDZ mediated interactions in PDZbase database involving domains considered in this work. The PWM scores have been computed as described in Materials and Methods.(XLS)Click here for additional data file.Table S2List of C-terminal sequences with nsSNPs modifying one single amino acid and predicted to disrupt or create PDZ binding motifs.(XLS)Click here for additional data file.Table S3List of C-terminal sequences with nsSNPs resulting in STOP codons and predicted to disrupt or create PDZ binding motifs.(XLS)Click here for additional data file.


Elastomeric proteins have evolved independently multiple times through evolution. Produced as monomers, they self-assemble into polymeric structures that impart properties of stretch and recoil. They are composed of an alternating domain architecture of elastomeric domains interspersed with cross-linking elements. While the former provide the elasticity as well as help drive the assembly process, the latter serve to stabilise the polymer. Changes in the number and arrangement of the elastomeric and cross-linking regions have been shown to significantly impact their assembly and mechanical properties. However, to date, such studies are relatively limited. Here we present a theoretical study that examines the impact of domain architecture on polymer assembly and integrity. At the core of this study is a novel simulation environment that uses a model of diffusion limited aggregation to simulate the self-assembly of rod-like particles with alternating domain architectures. Applying the model to different domain architectures, we generate a variety of aggregates which are subsequently analysed by graph-theoretic metrics to predict their structural integrity. Our results show that the relative length and number of elastomeric and cross-linking domains can significantly impact the morphology and structural integrity of the resultant polymeric structure. For example, the most highly connected polymers were those constructed from asymmetric rods consisting of relatively large cross-linking elements interspersed with smaller elastomeric domains. In addition to providing insights into the evolution of elastomeric proteins, simulations such as those presented here may prove valuable for the tuneable design of new molecules that may be exploited as useful biomaterials. Elastomeric proteins such as elastin, resilin, abductin and wheat gluten represent a remarkable class of self-assembling proteins that provide properties of extensibility and elastic recoil. Although unrelated from an evolutionary viewpoint, these proteins nonetheless share a common sequence design involving highly repetitive elastomeric regions interspersed with elements capable of forming cross-links that help stabilize the formation of polymers. Attempts to explore the influence of domain architecture on the self-assembly and mechanical properties of elastomeric proteins at the molecular level have largely been hindered by a general lack of detailed structural information. Here we introduce a novel theoretical study based on random walks to simulate the self-assembly of elastomeric proteins. Applying this model, we explored the impact of different configurations of elastomeric and cross-linking elements on the stability of the resultant polymer. Through exploring the complex relationships between elastomeric domains, required to drive self-assembly, and cross-linking domains, required for structural integrity, results from these simulations provide insights into the molecular basis for the evolution of elastomeric proteins as well as help guide the rational design of novel elastomeric-peptides. Elastomeric proteins such as elastin, resilin, abductin, spider dragline silks and wheat gluten represent a remarkable class of self-assembling proteins that provide properties of extensibility and elastic recoil in vitro in a reversible temperature-induced process termed coacervation, reliant on interactions between elastomeric domains In addition to imparting properties of elastic recoil, the hydrophobic regions of elastomeric proteins are thought to be responsible for driving their self-assembly at least for elastin For elastin, a number of molecular dynamic simulations have been performed in attempts to derive insights into the structural and biomechanical properties of elastin Here we describe a theoretical study that applies a modified Diffusion-Limited Aggregation (DLA) X. which describes the number of potential ‘surface moves’ a particle may make after aggregating and essentially reflects the relative diffusion speed compared to the speed of aggregation. The probability of a particle moving into the new site was based on the Boltzmann function:KT is a tunable temperature term, which effectively allows the particles to explore a greater number of conformations. This process is repeated several thousand times to construct large aggregates reminiscent of the fibrous-type structures associated with elastomeric fibrils We devised a modified implementation of an off-lattice DLA model to simulate the self-assembly of elastomeric molecules, represented by circular rods composed of elastomeric and cross-linking domains combined into a variety of different architectures. Previous work on elastin polypeptides have shown that they self-assemble into biomaterials upon changes in temperature KT and X in simulations of rods composed of a single elastomeric domain .In an initial series of simulations we first explore the impact of surface diffusion on generating compact fibres through varying parameters c domain . At low gregates . Such aggregates . As KT aXenopus can have as many as 48 domains Elastomeric proteins are composed of alternating ‘domains’ of elastomeric and cross-linking elements. It has been demonstrated that elastin-like polypeptides with as few as five domains possess the ability to self-assemble KT and X results in the generation of more compact aggregates . Only aggregates composed of the asymmetrical rod architectures 31 and 32 had consistently large numbers of nodes with either high node degrees (>6) or betweenness (>100). These results indicate that these latter two architectures form aggregates that may best withstand stress. However, as noted above, rather than individual topological properties contributing to aggregate resilience, the combination of such properties are likely to play a more important role, i.e. rods which have high betweenness values but are well connected to other rods, are more resistant to applied stress. In this study we sought to construct a simulation environment that can help us understand the impact of domain architecture on the morphology and structural integrity of polymers composed of elastomeric proteins. As we have shown here, the relative size of these domains may have a profound influence on the ability of these molecules to correctly align their cross-linking domains to help stabilize the resulting polymeric matrix. One relevant finding was that increasing the number of domains had little impact on aggregate morphology, but allowed the formation of additional cross-links. Elastomeric proteins have evolved multiple times independently within the eukaryotic lineage. Though they differ greatly in their structure and mechanical properties, they have converged on a distinctive sequence ‘style’, composed of alternating elastomeric and cross-linking domains. Interestingly, since arising in the vertebrate lineage, no short forms of elastin have been identified, varying from 15 elastomeric domains interspersed with 14 cross linking domains (encoded by 18 and 14 exons respectively) in human to 24 elastomeric domains interspersed with 24 cross linking domains (encoded by 35 and 24 exons respectively) in zebrafish tropoelastin-2 4  have higher stress and strain at break (0.23±0.08 MPa and 103±24% respectively) than polymers produced by EP20-24-24  (0.19±0.08 MPa and 86±42% respectively). From our simulations, these improved mechanical properties appear to have arisen through increasing the cross-linking capabilities of these proteins, while at the same time having little impact on overall fibre morphology.While the origins of these proteins are still unclear, it appears that after their first appearance, there was a rapid period of evolution resulting in the formation of longer forms of these proteins. For elastin, many domains are encoded by a single exon, suggesting that for this protein at least, elongation of the alternating domain architecture occurred though duplications of pairs of elastomeric and cross-linking domain-encoding exons Xenopus and zebrafish contain copies of elastin that in addition to having more domains, also tend to have larger elastomeric domains (up to 146 residues as encoded with exon 46 of tropolastin 2 of zebrafish) Xenopus and zebrafish to increase the number of cross-links, we speculate that their larger size may be a necessary adaptation for driving hydrophobic interactions under cold-blooded conditions, as suggested previously A further prediction from our model is that smaller elastomeric domains are required to help reduce the potential for misalignment between cross-linking domains on neighbouring rods. For human elastin, we note that elastomeric domains show relatively variable sizes, from 25 amino acid residues (encoded by exons 28 and 29) up to 54 residues (encoded by exon 20). Furthermore, During this work, we note that more resilient aggregates form when the monomer is composed of relatively large cross-linking domains interspersed with relatively short elastomeric domains. Such patterns help align neighbouring rods and thereby increase the probability of forming cross-links. However, in these simulations, we did not take into account the absolute size of overlap required for a rod to accrete to the growing aggregate, i.e. in nature it is likely that a minimum size of hydrophobic interface is required to drive assembly. Nonetheless, this study highlights the potentially complex relationship between the relative size of the elastomeric domain required for self-assembly, and the impact of its size on the ability of the resultant aggregate to form cross-links. From a biomaterials perspective, to generate polymers with greatest cross-linking potential it may therefore be important to design monomers with relatively small domains that are highly hydrophobic. On a related note, an interesting finding was that asymmetric rods resulted in the most resilient aggregates. Again this suggests additional criteria that bioengineers may wish to take into consideration in the design of novel biomaterials.in vivo. We note, for example, a very recent structural study that investigated tropoelastin by small angle X-ray and neutron scattering, which suggests that while tropoelastin is an elongated molecule, it is asymmetric, possessing a “foot”-like structure at one end It should be appreciated that in developing our modelling framework, we have made several assumptions. For example, we represent monomers as inflexible rods and introduce defined rules concerning the hydrophobic contribution to assembly as well as rules concerning the formation of cross-links. Such assumptions are likely to influence our findings and may not accurately reflect what occurs http://www.compsysbio.org/projects/rodDLA).This study raises several testable hypotheses concerning the evolution and design of elastomeric polymers (e.g. shorter elastomeric domains are predicted to result in aggregates with more cross-links) that may be readily tested in experimental systems that focus on the design of novel biomaterials based on spider silk and elastin proteins http://www.opengl.org). All software is made freely available under the open source software license at http://www.compsysbio.org/projects/rodDLA.Simulations and visualizations were performed using software developed in-house. The DLA simulator was written in C++ and was developed under Ubuntu Linux (version from 7.04–9.10). The 3D visualization tool was developed using QT (version 4.10 above) and OpenGL . To determine if the rotation is accepted, the total overlap of hydrophobic surface, using the distance threshold between rod axes of 1.2 units noted above, was calculated and used to assign a probability according to the formula based on the Boltzman function length is simply the length in units of the aggregate on the longest dimension. The connectivity of a rod is defined as the number of neighbours of the rod. Neighbours are defined as rods whose central axes are within 1.2 units of each other. Aggregate density is defined as the number of rods that transverse a defined cross section of length 10 units along the longest axis of the aggregate. The cross section is defined as a convex hull that encompass all the rods that transverse it, computed using Graham's Scan algorithm Surface area is approximated through dividing the aggregate into 10 unit sections along the longest axis of the aggregate. Perimeters of each section are then summed. The surface area is calculated only for the central section that occupies 50% of the aggregate's length. A cross-link between two rods is defined if the overlap between two neighbouring cross-linking domains is at least 50% of the length of both domains. Cross-linking networks are generated by treating rods as individual nodes. Edges between the nodes are then created if the rods share at least one cross-link. Betweenness, cluster coefficients, and node degree were computed using the BGL library in MatLab (http://www.mathworks.com/matlab).Aggregate Figure S1Impact of surface diffusion on aggregate morphology for a rod composed of multiple domains. (A) View of a section of an aggregate generated with rods composed of an alternating domain architecture of two cross-linking domains (red) and three elastomeric domains (blue). Each domain was four units in length and the rod had a diameter of one unit. The aggregate was constructed using KT = 20 and X = 1,000. (B) Phase diagram showing a two dimensional cross-section through a central 60 unit section of representative aggregates composed of multi-domain rods grown under different values of KT and X. (C) Side views of 80 units of the central section of representative fibrils from (B). (D) Quantitative measures of morphological characteristics of aggregates represented in the phase diagram (B). Aggregates were grown using 10,000 rods, standard deviations are from 10 replicates.(EPS)Click here for additional data file.Figure S2Quantitative measures of aggregate morphology and stability for 32 different rod architectures. Graphs showing measures of morphology and stability for aggregates generated for 32 different rod architectures composed of different numbers and sizes of elastomeric and cross-linking domains. Domain architectures are indicated at the bottom and are ordered according to increasing average node degree (e degree . Error b(EPS)Click here for additional data file.


Conserved domains are recognized as the building blocks of eukaryotic proteins. Domains showing a tendency to occur in diverse combinations (‘promiscuous’ domains) are involved in versatile architectures in proteins with different functions. Current models, based on global-level analyses of domain combinations in multiple genomes, have suggested that the propensity of some domains to associate with other domains in high-level architectures increases with organismal complexity. Alternative models using domain-based phylogenetic trees propose that domains have become promiscuous independently in different lineages through convergent evolution and are, thus, random with no functional or structural preferences. Here we test whether complex protein architectures have occurred by accretion from simpler systems and whether the appearance of multidomain combinations parallels organismal complexity. As a model, we analyze the modular evolution of the PWWP domain and ask whether its appearance in combinations with other domains into multidomain architectures is linked with the occurrence of more complex life-forms. Whether high-level combinations of domains are conserved and transmitted as stable units (cassettes) through evolution is examined in the genomes of plant or metazoan species selected for their established position in the evolution of the respective lineages.ATX1/2 and ATX3/4/5) have different phylogenetic origins. The two subgroups occur together in the earliest land plants Physcomitrella patens and Selaginella moellendorffii.Using the domain-tree approach, we analyze the evolutionary origins and distribution patterns of the promiscuous PWWP domain to understand the principles of its modular evolution and its existence in combination with other domains in higher-level protein architectures. We found that as a single module the PWWP domain occurs only in proteins with a limited, mainly, species-specific distribution. Earlier, it was suggested that domain promiscuity is a fast-changing (volatile) feature shaped by natural selection and that only a few domains retain their promiscuity status throughout evolution. In contrast, our data show that most of the multidomain PWWP combinations in extant multicellular organisms (humans or land plants) are present in their unicellular ancestral relatives suggesting they have been transmitted through evolution as conserved linear arrangements (‘cassettes’). Among the most interesting biologically relevant results is the finding that the genes of the two plant Trithorax family subgroups (O. tauri and in the metazoan lineage seems to have occurred independently consistent with convergent evolution. Our results do not support models wherein more complex protein architectures involving the PWWP domain occur with the appearance of more evolutionarily advanced life forms.Gain/loss of a single PWWP domain is observed throughout evolution reflecting dynamic lineage- or species-specific events. In contrast, higher-level protein architectures involving the PWWP domain have survived as stable arrangements driven by evolutionary descent. The association of PWWP domains with the DNA methyltransferases in  Conserved protein domains appear as a single (the only recognizable) architectural unit or in diverse combinations with a variety of other domains, a feature referred to as “domain versatility” or “promiscuity” -3. Two- first, whether the complexity of the domain architecture of chromatin proteins has increased during eukaryotic evolution; second, whether the assembly of a “versatile” conserved protein domain into multiple-domain architectures is linked to the occurrence of multicellular life-forms; and third, whether arrangements involving the PWWP domain represent ancestral versus reinvented architectures.Here, we use the domain-based tree approach to analyze the PWWP domain’s evolutionary patterns and to test these opposing hypotheses. The PWWP domain has been identified among the highly promiscuous domains present in proteins involved in various forms of signal transduction, in the ubiquitin system, and in chromatin remodeling ,12-15. TWHSC1 (Wolf-Hirschhorn Syndrome Candidate1) gene [The PWWP domain, discovered in the e1) gene  is namede1) gene -21. Emere1) gene , with DNe1) gene -27, or be1) gene .By sequence similarity, multiple alignment, and tree-reconstruction approaches, we investigate the phylogenetic relationships among the PWWP domains of proteins from the genomes of species with established evolutionary relationship within the plant and metazoan lineages  is among the earliest members of the green lineage [Chlamydomonas reinhardtii   and Volvox carteri  (Volvox) were chosen to follow the fate of the PWWP domain in connection with multicellular transitions [Physcomitrella patens  (Physcomitrella) and the lycophyte Selaginella moellendorffii  (Selaginella) are used as models for the early evolution of the PWWP  genes in land plants. Physcomitrella is considered a phylogenetic link half-way between algae and angiosperms [Sorghum bicolor was chosen as a model for a monocot because its genome is highly related to the other grasses, but contains fewer repetitive DNAs than its closest relatives [Populus trichocarpaArabidopsis thaliana and Arabidopsis lyrata were analyzed as eudicot models. The need to thrive in fixed locations over centuries under changing environmental conditions and biotic and abiotic stresses sets the poplar apart from the short-lived herbaceous plants. Populus and Arabidopsis lineages have diverged ~100–120 Ma and Populus homologs have been identified for each Arabidopsis gene, but poplar has more (1.4–1.6) protein-coding genes than Arabidopsis [The species with the smallest genome among the free-living eukaryotes  lineage . Here, insitions -35. The iosperms -38 whileiosperms -41. Sorgelatives . Since ielatives  facilitabidopsis . The conbidopsis .Homo sapiens and the cnidarian, sea anemone (Nematostella vectensis). The gene number and composition of Nematostella is more similar to vertebrates than are flies and nematodes suggesting that the complexity shared with humans must be ancient [PWWP  domain genes back to their unicellular origins, we analyzed the choanoflagelatum, Monosiga brevicollis  (Monosiga), sharing a unicellular ancestor with Metazoa more than 600 million years ago [The evolution of the PWWP-encoding genes in the animal lineage was followed in the genomes of  ancient . To tracears ago .Analyzing a limited selection of ancestrally related genomes positioned at important transitional stages in the evolution of either the plant or metazoan lineages allowed us to ask: 1) whether the modular evolution and the presence of the PWWP domain in complex protein architectures is linked to the occurrence of more complex life-forms; 2) whether assembled PWWP domain combinations are evolutionarily conserved and transmitted as cassettes, and 3) whether the PWWP domain has become promiscuous independently in different lineages through convergent evolution and, thus, the protein architectures are of multiple evolutionary origins and readily reinvented ., and Ostreococcus  domain is present in an SNF2 protein from Chlorella variabilis. The results suggest that some algal SNF2 and ASH1 proteins have ‘gained’ a PWWP domain as species-specific features.PWWP in multidomain combinations found in Volvox and in Chlamydomonas , but not in land plant relatives, are illustrated by two Volvox (XP_002950874 and XP_002951402) and two Chlamydomonas (XP_001692864 and XP_001701055) proteins in Figure Chlorella variabilis, Micromonas pusilla, O. lucimarinus and O. tauri). Interestingly, these proteins have no similar counterparts in the land plants but are closely related to bacterial proteins (without the PWWP domain) suggesting common origins. In the unicellular marine eukaryotes the PWWP domain has apparently been ‘gained’ for algal specific functions.Two solo PWWP domain containing proteins in Chlamydomonas  are highly related to the Volvox , proteins. Thereby, the O. tauri protein XP_003078017 represents an ATX3-architype and the earliest ATX3 predecessor (among available sequenced genomes). It carries all structural domains in a linear order conserved in land plants suggesting that it has been transmitted as a stable unit through evolution.A PWWP domain, associated with an ePHD, and SET domains, but lacking the DAST domain, is present also in i Figure e. PhylogO. tauri represents the earliest version of the ATX3/4/5 trithorax subtypes. These linear arrangements have been transmitted as stable, not volatile, combinations through green plants’ evolution. In addition to the conserved cassettes, more domains are present on the respective algal proteins  as a probe [A. thaliana  from orthologs (the members of the other clades) and for revealing correlations between whole-protein structures and their segregation into specific subgroups. Fourteen genes encode PWWP-containing proteins in A. lyrata  and the proliferation of their gene numbers imply strong functional relevance, although no function for any protein from this subgroup has been reported.ATM gene in the ancestor of the Arabidopsis lineage after its separation from the other plants. These proteins were not included in further tree reconstructions.Related to clade 1 are the PWWP domains of two proteins from the ATM (ATAXIA-TELANGIECTASIA MUTATED) family  domains [3Clades 2 and 3 are remarkable in that they contain the five proteins from the Arabidopsis Trithorax-like (ATX) family. The PWWP-domain based tree approach used here segregated the proteins into the same two subgroups established earlier by the relatedness of their SET , the meA. thaliana protein NP_197706, HUA2, contains a nuclear localization signal and may act as a transcription factor [HUA2 genetically facilitates the AGAMOUS, AG, gene [The last cluster (4) contains PWWP domains in combination with the VHS_ENTH_ANTH domains. The VHS, ENTH and ANTH domains are structurally similar, can bind inositol phospholipids, and are found in proteins involved in the nucleation and formation of clathrin-coated vesicles . The onln factor . HUA2 geAG, gene -59 but tP. trichocarpa, four in S. bicolor, six in A. thaliana, and four in A. lyrata encode the proteins in clade 1 of Figure S. bicolor are weakly related to the proteins of clade 1 . Four genes in Arabidopsis, three in elopment -59, it iO. tauri, and of ATX1, found in Chlamydomonas, illustrate the transmission of complex domain arrangements as stable units. Copies of these genes, appearing together for the first time in the moss, are present in all examined here land plants in species-specific copy numbers; 4) all PWWP domain cassettes found in land plants are found also in unicellular algae suggesting they have been inherited from the common ancestor. A notable exception are the PWWP- VHS_ENTH_ANTH containing proteins as no related proteins were found in the available genomes of unicellular organisms. These proteins may have occurred as a function specific for the land plants.Collectively, the analyses of the PWWP domain containing proteins in land plants indicate that: 1) all PWWP-containing proteins are phylogenetically related consistent with common ancestral origins; 2) the solo PWWP domain-containing proteins, only weakly related to the solo PWWP domain proteins of Chlamydomonas/Volvox, are the most abundant indicating a specific proliferation of this group in the land plants; 3) the ancestral version of ATX3, found in Monosiga brevicolis is considered the closest known relative of metazoans [M. brevicollis PWWP-domain proteins is shown in Figure NCBI database was revealed. Low-level similarity limited to the PWWP domains is displayed by the Monosiga protein XP_001747323 and the animal HDGF (Hepatoma derived growth factor), a nuclear protein with mitogenic activity [The unicellular choanoflagellate etazoans . Becauseactivity .M. brevicollis  proteins (XP_001747378) carries two PWWP domains, two HMG boxes, and three PHD fingers associated with AWS-SET domains as the earliest recognized version of the NSD (WHSC) family members  also belongs to the type ‘a’ and the presence of a PWWP domain in MSH6 is an exclusively metazoan feature, as a similar combination has not been found in any other mismatch repair in the examined genomes. The PWWP domain of the human GLYR1 (glyoxylate reductase 1 homolog of Arabidopsis) is phylogenetically related to the ‘b’-type. Despite highly similar to the plant glyoxylate reductase, only the metazoan proteins carry a PWWP domain. The cnidarian homologous proteins do not carry PWWP-domains suggesting that the PWWP-domain was gained after the metazoan divergence from Nematostella.Two cassettes containing a PWWP-PHD and BROMO domains are found in the ZMYND8/11 and Peregrin (BRPF) families. Despite similar modular structures, the PWWP domains from the two cassettes distribute into different subgroups  the combinations of the PWWP domain with the mismatch repair function (in HSU54777 protein), and with the glyoxylate reductase 1 homolog (in the GLYR1 protein) appear to be metazoan-specific.Collectively, our analyses of the PWWP domain containing proteins in the metazoan lineage suggested that: 1) the higher-level architectural PWWP assemblies found in the unicellular n Figure a is founn Figure ; the genn Figure ; 3) the The evolution of complex protein architectures has been linked to the requirements for novel functions during the occurrence of multicellular life-forms ,2. Here,vice versa. Thereby, a characteristic feature of single-PWWP domain proteins is that they are of different phylogenetic origins and with limited distribution among closely related species. Examples are the single PWWP domain proteins of M. brevicolis and from Chlamydomonas/Volvox that have homologs in other marine metagenomes but are not conserved in the animal or plant lineages. Interestingly, the existence of highly similar proteins of bacterial origin, but lacking the PWWP domain, indicate that marine unicellular eukaryotes have ‘gained’ a PWWP domain for algal specific functions. No solo PWWP domain encoding genes were found in O. tauri  database. Three search methods were used to mine new PWWP-domain proteins from all listed organisms: BLAST protein similarity searches were conducted by BLASTP [http://www.phytozome.net/), as well as from the DOE Joint Genome Institute (http://www.jgi.doe.gov/). After performing the similarity searches, all non-redundant hits were compiled and each of these sequences was manually examined to confirm the presence of the PWWP domain by searching the Conserved Domain Database (CDD) available from NCBI [http://www.ebi.ac.uk/Tools/pfa/iprscan/) was used. All 14 applications, including TIGRFAM, Superfamily, Gene3D, Panther, PFam, were used to re-test and confirm the analyses. In general, there was a very good consistence between the various prediction models from these applications and those at NCBI.PWWP-domain sequences were initially searched in y BLASTP  using thy BLASTP  was usedy BLASTP . Each qurom NCBI  as well rom NCBI ,76. In aThe ClustalX software, version 2.0  was emplNeighbor-Joining phylogeny of PWWP containing proteins was conducted with the MEGA4 program . The evohttp://phylogeny.lirmm.fr/[Maximum Likelihood phylogeny of PWWP containing proteins was performed with the “Phylogeny Pipeline” at lirmm.fr/. Multipllirmm.fr/, curatiolirmm.fr/, Maximumlirmm.fr/,85, and lirmm.fr/.RAV performed sequence and phylogenetic analyses, as well as tree constructions; ZA conceived the study and analyzed the data. Both authors contributed to the writing and preparation of the manuscript. Twelve genomes used in this study.Click here for file Phylogeny of the 12 taxa included in the study.Click here for file The genes encoding the PWWP domain containing proteins in A. thaliana and in A. lyrata.Click here for file Maximum Likelihood phylogeny of PWWP containing proteins in A. thaliana. Click here for file Neighbor-Joining phylogeny of PWWP containing proteins in A. thaliana. Click here for file Multiple sequence alignments used for generating the phylogenetic trees in this study.Click here for file Gene architecture of the PWWP domain containing proteins from the 12 genomes studied.Click here for file Neighbor-Joining phylogeny of PWWP containing proteins in plants including the ATX3-like protein fromO. tauri and the ATX1-like protein of Chlamydomonas.Click here for file Neighbor-Joining phylogeny of PWWP containing proteins in humans, Nematostella, M. brevicolis and O. tauri .Click here for file


Many protein-protein interactions are mediated by domain-motif interaction, where a domain in one protein binds a short linear motif in its interacting partner. Such interactions are often involved in key cellular processes, necessitating their tight regulation. A common strategy of the cell to control protein function and interaction is by post-translational modifications of specific residues, especially phosphorylation. Indeed, there are motifs, such as SH2-binding motifs, in which motif phosphorylation is required for the domain-motif interaction. On the contrary, there are other examples where motif phosphorylation prevents the domain-motif interaction. Here we present a large-scale integrative analysis of experimental human data of domain-motif interactions and phosphorylation events, demonstrating an intriguing coupling between the two. We report such coupling for SH3, PDZ, SH2 and WW domains, where residue phosphorylation within or next to the motif is implied to be associated with switching on or off domain binding. For domains that require motif phosphorylation for binding, such as SH2 domains, we found coupled phosphorylation events other than the ones required for domain binding. Furthermore, we show that phosphorylation might function as a double switch, concurrently enabling interaction of the motif with one domain and disabling interaction with another domain. Evolutionary analysis shows that co-evolution of the motif and the proximal residues capable of phosphorylation predominates over other evolutionary scenarios, in which the motif appeared before the potentially phosphorylated residue, or vice versa. Our findings provide strengthening evidence for coupled interaction-regulation units, defined by a domain-binding motif and a phosphorylated residue. Domain-motif interactions are instrumental for many central cellular processes, and are therefore tightly regulated. Phosphorylation events are known modulators of protein-protein interactions in general, including domain-motif interactions. Here, we addressed the association of phosphorylation and domain-motif interaction taking a motif-centred view. We integrated human domain-motif interaction and phosphorylation data for four representative domains , and showed that the adjacency between phosphorylation and domain-motif interactions is extensive, suggesting interesting functional links between them that extend the classical and widely studied phospho-regulation of SH2 or WW domain-motif interactions. Furthermore, we show that such interaction-regulation units may function as double switches, concurrently enabling interaction of the motif with one domain and disabling interaction with another domain. These latter interaction-regulation units are more conserved in evolution than the individual units comprising them. Assuming that the four analyzed domain-motif interaction types are reliable representatives of such interactions, our results support the existence of units comprising motifs and associated phosphorylation sites, in which the regulation of domain-motif interaction is inherent. Hence, deciphering the basic details of this network, the interacting protein pairs and the protein elements mediating the interaction, is a major challenge. In the last decade it became widely accepted that protein domains play a key role in mediating protein-protein interactions. A prominent type of domain-mediated protein-protein interaction is domain-motif interaction, commonly achieved by a domain in one protein and a short linear motif in the interacting partner e.g. interactions between SH3 domains and proline-rich motifs The e.g. SH2 and class IV WW domains e.g. SH3 and PDZ domains). The phospho-regulation of the former has been studied extensively while the phospho-regulation of the latter has been noted in sporadic cases. For instance, the interaction between NCK and PAK1, which is mediated by SH3-motif interaction, is prevented by phosphorylation of a residue just near the motif The comprehensive involvement of domain-motif interactions in key cellular processes necessitates tight regulation. Protein phosphorylation is well-accepted as a generic regulator of protein-protein interactions, including domain-motif interactions Here we study this regulatory mechanism, focusing especially on motifs in which phosphorylation is not required for domain binding, but rather might play a preventive role. The results of our large-scale integrative study point to the existence of coupled interaction-regulation units, where phosphorylation within or near the motif is suggested to play a role as an ‘on’/‘off’ switch of domain-motif interactions.We chose human as the organism for our study, due to the wealth of phosphorylation and domain-motif interaction experimental data. First, we generated a comprehensive database of protein phosphorylation events derived from nine data sources , where we.g. a database curator will naturally find one reference with a documented motif that binds an SH2 domain, along with the relevant phospho-tyrosine), we repeated our randomization test using only high-throughput phosphorylation data and low-throughput motif data. In the majority of the cases, the association between motifs and phosphorylation sites remained statistically significant  or domains of the same family (section II below).e.g. YxNx pattern) and an SH3- binding motif (e.g. PxxDY pattern) i.e. based on low-throughput methods). These results strengthen the conjecture that motif phosphorylation may function as a double switch for binding domains from different families.Consider the following scenario: a protein segment includes two merged motifs: an SH2-binding motif .We followed the evolutionary history of the interaction-regulation unit components by examining the human motif sequences and phosphorylation sites in orthologous proteins present in 15 eukaryotic organisms. We focused on human proteins that include units with motifs that match a previously characterized sequence pattern see . Each su2 test, see Our comparative analysis, applied to an established eukaryotic phylogenetic tree X. Tropicalis  that is phosphorylated in human, leading to trivial co-appearance of the interaction-regulation unit components. To circumvent this potential bias, we repeated the analysis using only ELM-based regular expressions alis see . Thus, mDomain-motif interactions are instrumental for many central cellular processes, and are therefore tightly regulated. Phosphorylation events are known modulators of protein-protein interactions in general, including domain-motif interactions. The association between domain-motif interactions and phosphorylation events may stem from their similar interaction time scales (kinase-substrate interactions are themselves domain-motif interactions). Phospho-regulation of domain-motif interaction is apparent in cases where the motif-binding cleft in the domain is phosphorylated, resulting in loss of its interaction potential . This dual motif may suggest an alternative ABL2 regulation via on/off switching of self-interaction with its own SH3 and SH2 domains by Tyr-568 phosphorylation. Several lines of evidence strengthen this hypothesis: First, the corresponding tyrosine in mouse ABL-2 is documented as being phosphorylated One intriguing candidate for SH3/SH2 phospho-switching is a dual motif, e.g. through kinase/phosphatase levels).Our results strengthen phosphorylation/de-phosphorylation double switch of protein-protein interactions as an important control mechanism among other widespread regulation schemes, mostly related to the cellular context of the proteins . Cellular context may also affect phospho-switching of domain-motif interactions , should be investigated for both their positive and negative effects, such as enhancement or prevention of interactions.Concurrent enabling and prevention of interaction involving domains from the same family may be static rather than temporal. For example, following our paper Our analysis revealed also a tight coupling between motifs and phosphorylation sites in their flanking 20 residues in both sides. This context length is in accord with previous publications studying the same domain-motif interaction types Phosphorylated residues near a domain-binding motif may affect the domain binding, as shown for the interaction between nuclear localization signal motifs and ARM domains e.g. phosphorylation event) may affect the motif's binding-related behaviour.The protein segment that includes the interaction-regulation unit is bound by multiple proteins: the kinase, the corresponding domain, and, in case of a double switch, a second domain. Thus, this protein region needs to adopt different conformations upon interaction with two or three distinct domains. This flexibility is probably feasible due to the tendency of phosphorylation sites and short motifs to reside in disordered regions . Proteinet al.We conducted sequence comparison between human and several eukaryotic organisms to trace the path in which the interaction-regulation unit components appeared during evolution. In 46% of the cases, the unit's components probably appeared together, frequently in vertebrates, and remained conserved along this lineage. Possibly, the co-appearance of the interaction-regulation unit components may trivially result from a protein that appeared in a certain phylogenetic branch and remained highly conserved up to human. Notably, in 77% of the cases where the unit components co-appeared, the protein that includes the unit has an ortholog in more distant organisms, but their sequence does not include the corresponding unit's region. This implies that, in general, the motif appears in evolution along with the potential to be phospho-regulated. This also agrees with the results of Chica We identified also step-wise paths for the appearance of the interaction-regulation unit components. First, there are cases in which the motif probably appeared without being phospho-regulated. Such regulation appeared later, perhaps in cases where the domain-motif interaction required tighter regulation. The second evolutionary trace regards an early appearance of the potential phosphorylation site, followed by the motif's appearance. This scenario might be explained by an ancient functional phosphorylation event that has the potential to induce a conformational switch that exposes its nearby protein environment. This switch was later ‘hijacked’ by a newly introduced domain-binding motif that exploited it for its own regulation. The ‘early phosphorylation’ and the ‘early motif’ evolutionary traces cover 20% and 34%, respectively, out of the 481 studied evolutionary traces.et al.Conservation of phosphorylation was recently studied by Tan Our findings have important implications for elucidating the function of motifs and phosphorylation events. The abundance of motif phospho-regulation implies that the search for novel domain-binding motifs should be followed by searching for intra/near phosphorylation sites. Similarly, newly discovered phosphorylation sites should be checked for a nearby binding motif, which may shed light on their function. The evolutionary trace of the motif and its flanking regions should assist in this regard. Another promising direction is towards a comprehensive protein-protein interaction network connecting between the interaction-regulation units, their corresponding kinases and the domain-containing proteins interacting with them. Analyzing this network should reveal novel associations between kinases and domain-motif interactions.e.g. phospho-specific antibodies), and (b) ‘high throughput’, based on large-scale experiments (mostly mass-spectrometry).We integrated six databases of human experimentally-verified protein phosphorylation sites , we chose only one domain-motif interaction (preferably the shorter motif). We focused on SH2, SH3, PDZ and WW domain-motif interactions, for which the largest amounts of data were available. This catalogue of 1,983 interactions was divided into two datasets: (a) ‘low throughput’, highly reliable data including 867 interactions based on low throughput experiments . (b) ‘high throughput’, including 1,116 interactions based on large scale experiments (e.g. protein micro-arrays). To compute the number of experimentally verified motifs that include a previously characterized sequence pattern, we used several repositories of such sequence patterns: Scansite We extracted human domain-motif interactions from seven databases PepCyber  and inteTo assess the statistical significance of our findings we compared the motif-phosphorylation coupling found in the data to that found in randomized datasets. For each protein that includes a motif of a certain type, we selected randomly same-length sub-sequences along the protein sequence while keeping the phosphorylation positions fixed. These random sequences obeyed the following constraints: (a) The random motif did not overlap with the actual motif and included a Ser/Thr/Tyr residue. (b) Since motifs and phosphorylated residues are known to reside within disordered protein regions S. cerevisiae, we integrated high-throughput domain-motif interactions from Landgraf et al.To create a database of motifs and phosphorylation sites in a interacts with a non-phosphorylated peptide, but not with the ‘pseudo-phosphorylated’ peptide, and PDZb displays an inverse binding pattern. We identified 81 peptide-PDZ double pairs that showed this binding pattern . These PDZ pairs were used to evaluate the statistical significance of the results. In principle, given a non-phosphorylated/pseudo-phosphorylated peptide pair and two PDZ domains there are 10 possible scenarios of binding/non-binding relationships among them . The conservation level of human motifs and phosphorylation sites were deduced from pair-wise sequence alignment between the human protein and each relevant ortholog. We chose a pair-wise, rather than multiple sequence alignment approach, since the multiple sequence alignment scheme produced mis-aligned segments of ortholog sequences. This was probably due to the fact that the differences between the orthologs were not consistent (in terms of substitutions and insertions/deletions). The organism that is most distant from human and has a Ser/Thr/Tyr residue in a corresponding position was set as the organism in which the potential phosphorylation site first appeared. Likewise, we determined the organism in which the motif appeared. Here, we searched for a respective sequence pattern hit by the four servers mentioned above. Since the location of motifs in orthologs was previously shown to be flexible, we considered motif appearance if it was found within 20 residues range  of the sequence that aligned with the human motif. Note that the different pattern recognition tools identify different subtypes of the motif. We unified these different types and treated them as one. In the evolutionary analysis of the SH2- and class IV WW-motifs, we disregarded the phosphorylation sites that are essential for the interaction. For this analysis we used both the low throughput and the high throughput datasets of the human domain-motif interactions and phosphorylation events.The set of human motifs that were used for the evolutionary analysis was restricted to motifs that obey a previously characterized sequence pattern. For SH3, SH2 and PDZ motifs, we used ScanSite prediction 2 test Click here for additional data file.Figure S2Phylogenetic traces of interaction-regulation unit evolution.(PDF)Click here for additional data file.Table S1Counts and statistical significance of the coupling between domain-motif interactions and phosphorylations.(PDF)Click here for additional data file.Table S2Identification of human protein segments that display a dual motif that can act as a phosphorylation double switch for SH3 and SH2 domains or for SH2 and WW domains.(PDF)Click here for additional data file.Table S3Proteins that are predicted to be involved in ‘double switches’ based on experimental protein-protein interaction data.(PDF)Click here for additional data file.Table S4Phosphorylation specificity switch for proteins containing domains of the PDZ family.(PDF)Click here for additional data file.Table S5Statistical significance for the PDZ double-switch analysis.(PDF)Click here for additional data file.Table S6Statistical significance for the evolutionary path analysis.(PDF)Click here for additional data file.Table S7Disorder levels of the various motifs.(PDF)Click here for additional data file.Dataset S1Database of interaction-regulation units for Homo sapiens.(XLS)Click here for additional data file.Dataset S2Database of interaction-regulation units for Saccharomyces cerevisiae.(XLS)Click here for additional data file.


Schistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored.Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays.We present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system.SjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the S. japonicum. The SjGIPC3-PDZ domain is able to bind both type I and II ligand C-terminal motifs. The identification of native ligand will help reveal the potential biological function of SjGIPC3. These data will facilitate the identification of novel drug targets against S. japonicum infections.In this study, we first characterized a PDZ domain-containing protein GIPC3 in  Schistosoma, remains a major global public health problem that affects more than 230 million people living in the endemic areas of 77 countries worldwide (more than 95% in Africa) (http://www.who.int/mediacentre/factsheets/fs115/en/index.html). Three major pathogenic schistosome species, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum, are known to contribute to the disease loads. The treatment of schistosomiasis is based on the long-term mass treatment with the only available drug, praziquantel (PZQ). However, concerns about the problem of drug resistance have necessitated a search for alternative treatments -XΦ*; and Class IV, -Xψ-[D/E]*; where X is any amino acid, Φ is a hydrophobic residue, ψ is an aromatic residue, and * represents the stop codon of the peptide -[STIL]-[HSNQDE]-[VIL]* as bait; 92 proteins that physically fit the consensus binding sequence were fished out. We also performed BLAST searching on NCBI within taxa 6182 directly; using each unique carboxy-terminal sequence (the extreme four amino acids) obtained from both Y2H assays as bait. A panel of proteins whose C-terminal tail matched the unique sequences was retrieved. The protein sequences retrieved from both bioinformatics pipelines were used to perform BLASTP searches on NCBI to manually filter those peptides lacking the C-terminus. We also used integrated biological information, such as transmembrane region prediction, potential molecular functions, and the C-terminal sequence consensus between S. japonicum and S. mansoni to determine the most promising candidate ligands. Finally, six proteins were selected as ligand candidates of SjGIPC3  than in the first assay (13.5%). Similarly, at P-3, 16 types of amino acids emerged in the first assay, while only eight kinds of amino acids were presented in the second assay. Also at this position, the occurrence of Glu was 50.0% when screened against the full-length SjGIPC3, twice the occurrence rate as when the SjGIPC3-PDZ domain was used as bait. Thus, the binding specificity screened using full-length SjGIPC3 as bait may be more authentic to its native status.Screening a highly diverse peptide library provided an opportunity to fish out the potential binding property of SjGIPC3. Whether the SjGIPC3-PDZ domain or full-length SjGIPC3 were used as bait, analogous binding specificities can be deduced from the unique C-terminal tails obtained from both Y2H assays. However, the GH1 and GH2 domains of SjGIPC3 may still affect the binding specificity of the PDZ domain to some degree, because the ligand motifs were more canonical when full-length SjGIPC3 was used as bait. For instance, at PX-S/T-XΦ*), II (−ΦXΦ*), and III (−XX-C*) binding motifs [Drosophila, and GLUT1CBP each interact with Myosin VI through an internal sequence within the cargo binding domain [-2[0[Previously, GIPCs have been shown to interact specifically with Class I  in the context of the parasite-host interaction. More importantly, the preferential expression of SjGIPC3 in male rather than female adult worms further hinted that the potential function of the protein was related to the processing of environmental information, as male adult worms interface with the host and female adult worms resided in the gynecophoral canal, more stimulating signal would be received by male adult worms [To adapt to the complexity of the life cycle, schistosomes are equipped with a complex nervous system to sense environmental signals . Transcrlt worms . GIPC3 mlt worms -50. Prevlt worms . IntriguS. japonicum. We defined the ligand binding specificity of the SjGIPC3-PDZ domain by screening a random peptide library constructed with human genomic DNA. The ligand binding ability of the SjGIPC3-PDZ domain is relatively extensive; it can bind both type I and type II ligand motifs, which hints at its potential to play multiple roles during the development of S. japonicum, especially in the adult stage. Our study also provided a practical method for predicting potential ligands of SjGIPC3. Ongoing studies to identify more ligands involved in signal transduction followed by improvement of the quality of the S. japonicum proteomic dataset will assist in the discovery of novel drug targets against the parasite.For the first time, we have presented the molecular characterization of the PDZ domain-containing protein GIPC3 from All procedures performed on animals within this study were conducted following animal husbandry guidelines of the Chinese Academy of Medical Sciences and with permission from the Experimental Animal Committee.The authors declare that they have no competing interests.YM, PC, and YG conceived and designed the experiments. YM, HH, SL, and PC performed the experiments. YM, PC, and YG analyzed the data. YM, PC, and YG wrote the manuscript. All authors read and approved the final version of the manuscript.Table S1. Primer sequences used in this study. This file contains the information about the primers used for qRT-PCR and molecular cloning.Click here for file


The oddball paradigm is widely applied to the investigation of cognitive function in neuroscience and in neuropsychiatry. Whether cortical oscillation in the resting state can predict the elicited oddball event-related potential (ERP) is still not clear. This study explored the relationship between resting electroencephalography (EEG) and oddball ERPs. The regional powers of 18 electrodes across delta, theta, alpha and beta frequencies were correlated with the amplitude and latency of N1, P2, N2 and P3 components of oddball ERPs. A multivariate analysis based on partial least squares (PLS) was applied to further examine the spatial pattern revealed by multiple correlations.Higher synchronization in the resting state, especially at the alpha spectrum, is associated with higher neural responsiveness and faster neural propagation, as indicated by the higher amplitude change of N1/N2 and shorter latency of P2. None of the resting quantitative EEG indices predict P3 latency and amplitude. The PLS analysis confirms that the resting cortical dynamics which explains N1/N2 amplitude and P2 latency does not show regional specificity, indicating a global property of the brain.This study differs from previous approaches by relating dynamics in the resting state to neural responsiveness in the activation state. Our analyses suggest that the neural characteristics carried by resting brain dynamics modulate the earlier/automatic stage of target detection. The oddball task is a broadly applied paradigm in the investigation of cognitive function. Target stimuli are presented amongst more frequent standard background stimuli. A distracter stimulus may also be used to ensure that the response is to the target rather than to the change from a background pattern. The waveform of elicited evoked-potential is thought to reflect central electrical activity related to a number of cognitive processes, such as attention allocation, stimulus evaluation and memory-based comparison, and has been studied in a variety of neurological and psychiatric disorders -9. OddbaThe early (N1 and P2) components of oddball ERPs have been regarded to indicate automatic stimulus processing. They are influenced by early aspects of attention and orientation ,14. The Among the modulators of oddball ERPs, the perspective of neural-neural interaction has been less addressed in the previous literature. Nevertheless, neural-neural interaction is critical in the theoretical framework to explain the generation of ERP. For example, previous studies have proposed that phase-resetting and phase synchronization between different frequency bands play a substantial role in the construction of early ERP ,34. BarrThis study extends the previous understanding by incorporating resting brain dynamics into the study of auditory oddball ERPs. The resting neural informatics are actually irrelevant to the occurrence of oddball events, very different from previous approaches addressing brain dynamics at or after the occurrence of stimulus -35. We aA total of 233 right-handed healthy young females, aged 19-22, were enrolled. All the participants were evaluated by licensed psychiatrists following a semi-structured interview process. The neurological and physical examinations were performed by licensed medical doctors. Those who had a history of substance abuse, psychiatric disease, or major medical or neurological disorder were excluded. Only those who had been medication-free (including birth control pills) for at least two weeks were enlisted. 218 participants were included in the final analysis. This project was approved by the local ethics committee conforming with Helsinki declaration. Informed consent was obtained from all participants prior to the commencement of the investigation.All participants received a 3-minute conventional, eyes-closed, awake, digital EEG after a 5-minute habituation to the experimental environment . Recordings were in accord with the international 10-20 system, with linked ear reference, a 128 Hz sampling rate and impedance below 3 kΩ . The artAn auditory oddball paradigm was used to elicit the oddball event-related potential (ERP). Fifty target tones  and 275 non-target tones  were interspersed in random order and presented binaurally. The tone pips were delivered at a stimulation rate of 1.3 tones/sec, with a 50-ms duration for each . Subjects were required to count target stimuli while ignoring the frequent low-pitched tones. The data was discarded if the reported target number deviated more than 5 from the correct answer. The inter-trial interval of the auditory stimuli was varied in a 1,100-1,500 ms range  to avoid expectancy effect. Other oddball ERPs detail can be referred to elsewhere .2). Eighteen electrodes, including F7, F3, Fz, F4, F8, T3, C3, Cz, C4, T4, T5, P3, Pz, P4, T6, O1, Oz and O2, were included for further analysis with oddball ERPs.As to the resting EEG signals, Fast Fourier Transform (FFT) was applied to consecutively non-overlapped and artifact-free segments of 20 sec to derive the mean EEG power for each electrode at a specified frequency band  1/n, where n equals the number of comparisons. The statistical threshold of the Bonferroni correction is 0.0028 (n = 18). Given that the electro-cortical activities are not completely independent, we report both P < 0.01 and P < 0.0028 in case the multiple comparison correction is too stringent.This study aims at investigating whether the quantitative EEG indices in the resting state predict oddball ERPs parameters. For the oddball waveforms at Fz, Cz and Pz, the amplitude and latency of N1, P2, N2 and P3 were quantified. Our preliminary analyses revealed a highly correlated pattern across Fz-oddball, Cz-oddball and Pz-oddball. We thus performed principal component analysis (PCA) to reduce the three oddball ERPs to a representative one, PCA-oddball. The amplitude and latency of N1, P2, N2 and P3 from the extracted PCA-oddball were then correlated (Pearson's correlation) with resting state spectral powers. For each test set in this study, the criterion for significance is thresholded at For the correlation maps of resting dynamics and PCA-oddball showing significance, we used a multivariate method of partial least squares (PLS) to examine the spatial pattern of resting neuro-dynamics that predicted PCA-oddball. The PLS method was developed by McIntosh et al. and has been successfully applied in various neuroimaging studies, including EEG -63. PLS The grand mean waveform of oddball ERPs and resting EEG spectrum are illustrated in Figure N1 and N2 amplitudes are negatively correlated with regional power at the alpha band. Since we preserve the signs of negative peaks, our finding implies that the stronger the regional power at alpha in resting state, the greater the magnitude of the negative peaks at N1 and N2. The electrodes with significant correlations cover widespread brain regions, including frontal, temporal and parietal areas. The results of N1 and N2 amplitudes with regional power are summarized in Table http://www.websdj.idv.tw/kiki/rEEG_P300.pdf). The supplementary material also contains the results of multiple regression analysis with oddball ERPs and oscillatory powers as dependent and explanatory variables, respectively (Table S3).The latency of P2 is negatively correlated with the regional power at resting alpha band. This finding implies that stronger regional power at resting alpha would shorten the P2 latency. As in the analyses of N1/N2 amplitudes, most of the electrodes reveal significant correlations, indicating that the relationship is substantiated by a "global" neural characteristic, instead of regional specificity. The statistical threshold of the Bonferroni correction is the same as before, 0.0028. The results of the correlation of regional powers and P2 latency are displayed in Table Since the correlation maps at alpha frequency reveal a distributed, in opposition to focal, relation with N1/N2 amplitudes and P2 latency, associated spatial patterns are investigated using PLS. The PLS analyses comprise 2 categories of calculation, namely resting alpha-oddball amplitude and resting alpha-oddball latency. We notice that for each of the 2 PLS computations, only 1 prominent singular value satisfies the 10 percent criterion, with respective contribution 95.0 percent and 91.7 percent for oddball amplitude and latency. The permutation tests show that the respective probabilities to have singular values greater than the largest singular value are 0 and 0.0016. Concordant with the correlation maps, the saliencies associated with N1/N2 amplitudes as well as P2 latency are equipped with robust statistics, summarized in Table The oddball paradigm is a cognitive surrogate in probing the capability of target detection, which demands attention allocation, stimulus evaluation and comparison, and has been widely applied in a variety of neuro-psychiatric conditions. This study examines the hypothesis of whether the brain oscillation in the resting state, i.e. irrelevant to the active experiment, could predict the elicited oddball ERPs. Our design is different from that of event-related synchronization/desynchronization, which follows a triggering stimulus. Our analyses show that alpha power in the resting state is negatively correlated with the negativity of N1/N2 and the latency of P2. Without exception, the correlating trend (positive or negative) is consistent for all the significant qEEG indices in terms of each oddball ERPs component. The demonstrated aggregated correlation patterns indicate that the interaction between the resting versus activation conditions possesses spectral specificity. The finding of resting alpha oscillation and N1/N2/P2 is concordant with previous literature addressing the contribution of alpha dynamics to the generation of ERPs ,34. We dN1 and P2 have been associated with lower-level perceptual processing and mental speed, while N2 is relevant to short-term memory and influenced by stimulus novelty ,66. Our Stronger regional power at alpha is observed to shorten the P2 latency. As with the widely distributed alpha oscillations in predicting N1/N2 amplitudes, the significant correlations spread over most of the electrodes, again indicating that the relation of resting and activation conditions is substantiated by a "global" neural characteristic. This observation is further endorsed by the PLS computation. The oddball P2 was reported to be correlated with response time ,68,69, cEven though all oddball ERPs components are relevant to the processing of the target, the components P2 and N2 have been regarded as different from P3. It was reported that the P2/N2 stage reflected automatic processing, whereas the P3 stage was capacity-limited, as suggested by the design manipulating task difficulty . CoupledOur finding at alpha frequency is intriguing since alpha oscillation has been related to a wide variety of cognitive processes. For example, alpha frequency was significantly correlated with the speed of information processing  and alphIt has been suggested that ERP may be explained by superimposed oscillatory components, notably delta and theta components in oddball ERPs -85. The It is noteworthy that our findings do not provide an exclusive account for the oddball ERPs. In fact, there are plenty of psychological and biological factors that modulate the oddball ERPs profiles. For example, it was suggested that N1 amplitude was accentuated in response to enhanced cortical arousal and increased top-down cognitive effort -23, and Several studies have demonstrated that the baseline features immediately preceding stimulus onset can predict subsequent neural response or neuropsychological performance -94. It sThere are plenty biological and psychological modulators of oddball ERPs. The neuromatrix in the resting state has been reported to carry abundant information as an endo-phenotype. This study relates the brain dynamics in resting state to those in activation state of target detection. Our analyses suggest that the neural characteristics carried by resting brain modulate the earlier/automatic stage of target detection. The modulatory effects show spectral specificity at alpha band but no topographical preference.The authors declare that they have no competing interests.All the authors contributed to the conception and design of this project. Authors TJC and YWYY designed the study and refined the protocol. Author TWL managed the literature search and statistical analysis, and wrote the first draft of the manuscript. Authors TJC, YWYY and HCW executed the experiment protocol and undertook the EEG data collecting procedure, subject evaluation and quality control. All authors contributed to and have approved the final manuscript.