Streptococcus strains and studied metabolic diversity from a protein domain perspective. Metabolic pathways were described in terms of the promiscuity of domains participating in metabolic pathways that were inferred to be functional. Promiscuity was defined by adapting existing measures based on domain abundance and versatility. The approach proved to be successful in capturing bacterial metabolic flexibility and species diversity, indicating that it can be described in terms of reuse and sharing functional domains in different proteins involved in metabolic activity. Additionally, we showed striking differences among metabolic organisation of the pathogenic serotype 2 Streptococcus suis and other strains.Understanding the diversity and robustness of the metabolism of bacteria is fundamental for understanding how bacteria evolve and adapt to different environments. In this study, we characterised 121  Understanding the diversity and robustness of the metabolism of bacteria is a fundamental step in the study of organisms, as these may mirror the large variety of approaches and strategies that species have taken to adapt to the environment. Nonetheless, even in well-studied bacterial model organisms, our knowledge of metabolism is far from complete . SeveralThe prevailing paradigm is that new metabolic pathways, and thus new metabolic functions, arise through the exchange of enzymes from other existing metabolic networks . Enzyme In this study, we set out to characterise bacterial metabolic diversity—that is, the ability of gaining (or losing) new metabolic functionalities from a protein domain perspective, investigating the working hypothesis that can explain, or at least describe, the observed metabolic diversity in terms of reuse and sharing functional domains in different proteins.Functional domains can be defined as compact modules, characterised by structural and/or sequence conservation, folding from a single polypeptide chain . The aggThe underlying premise is that proteins were able to evolve into the flexible multifunctional entities that we know today by altering a protein’s domain architecture, either via gaining, losing, duplicating and substituting domains . This reStreptococci are a particularly good study target; several streptococcal species are non-pathogenic and are active members of the commensal human microbiome  sho sho42] sπ for domain d in a given strain s is given by the product of IAF, IV and α:π is an inverse measure of promiscuity, thus the smaller the π, the larger the promiscuity of a domain.Finally, the promiscuity score π can assume different values for the same domain when different strains are considered. This is a combined effect of the fact that the same domain can have a different number of neighbouring partners in different organisms and that the same domains can appear in a different number of pathways in different organisms. When the aim is to arrive at an absolute measure of promiscuity, a measure independent from the strain considered, we take a limiting approach, considering promiscuity as an ensemble property of a domain. For a given domain d, its absolute promiscuity π(d) is calculated asn is the number of strains used for calculation of the promiscuity. The limit in d is calculated considering 1, 2, 3, and so on, strains until convergence. The limiting procedure was realised by resampling 100 strains for n = 1, 2, …, 121.From Eqs t-dimensional binary vector Bs where t is the total number of domains inferred by considering all 121 strains, defined asB-vectors were then collapsed into a 121 × t metabolic matrix M for further analysis.For every strain, each metabolic pathway was vectorised to a Bs was mapped to a weighted metabolic promiscuity vector Ws simply defined asπ(d) and Bs(d) have been defined in Eqs Second, each element of x and y given by D is obtained as proposed in previous research [D, which is based on the cosine distance between two vectors, ranges from 0 to 1; 0 indicates that the two metabolic pathways share the same domains (each with the same promiscuity) while 1 indicates that no domains are shared between the two pathways. We only consider pathways that have at least 66% of their reactions performed by a protein assigned by Pathway Tools.Given two metabolic pathways their vectorised representations research :D=Streptococcus strains calculated using Sk for the k-th group can be written asXk is a matrix of standardised coordinates of the objects for the k-th matrix and Mk is a diagonal matrix of weights accorded by matrix k to each of its dimensions. Each matrix can be seen as a set of variables (the columns of Xk) defined on the objects. With this notation, the INDSCAL model can be written asA the M × R matrix of the coordinates of the objects equal for all the matrix (A is the loadings matrix) in a space with R dimensions and Wk is R × R diagonal matrix of the weights of matrix k. The elements on the diagonal of Wk are the coordinates for the k-th matrix in the R-dimensional space defined by INDSCAL model.INDSCAL  was usedR-dimensional space to highlight differences and similarities. We refer the reader to previous research [In this way, the different matrices can be then represented in an research ,44 for mresearch . The INDex-novo the 121 genomes to guarantee a uniform up-to-date annotation by applying the workflow depicted in Public gene databases are growing exponentially and double in size approximately every 10 months , implyinStreptococcus. Of the inferred pathways, 27 appear in all strains. Streptococcus strains considered. A complete list of all of the inferred pathways is given in Pathway reconstruction based on the metabolic reaction assignment by Pathway Tools resulted in 298 pathways available to Streptococcus strains. Several pathways are involved in the biosynthesis of ATP, GTP, UTP, S-adenosyl-L-methionine and amino-acid conversions from one amino acid to another, which are the basic needs of any living organism. Other core pathways are involved in the metabolism of the compounds related to energy, carbon and nitrogen source utilisation, such as degradation of mannose and fermentation of pyruvate. Finally, some core pathways pertain to the production of cell wall compounds, such as fatty acids, peptidoglycans and lipopolysaccharides.Those core pathways are involved in base cofactor production, base metabolism and cell wall/membrane metabolism: these functionalities constitute the base of bacterial metabolism and, not surprisingly, are found in all pyogenes: none of the 19 strains have the metabolic capacity to perform Glutamate degradation I, Glutamate degradation X, Glutamate biosynthesis II and Glutamate biosynthesis III, in comparison to other Streptococcus species where these pathways are always present. This finding is consistent with results from metabolic modelling [S. pyogenes to ferment glutamate [Streptoccus species [pyogenes does not ferment glutamate [pneumonia strains are the only Streptococci equipped with homospermidine biosynthesis and pyruvate oxidation pathways, which have been found to be involved in pathogenicity mechanisms [A striking example of missing metabolic activity is found in the species S. odelling  and withlutamate : this in species , and it lutamate  despite chanisms  and in tchanisms .Streptococcus strains and the 298 active metabolic pathways, we obtained a collection of 881 functional domains. A complete list of the domains with the associated InterPro identifier is given in Functional domains were inferred using the domains of the gene products active in the metabolic reactions, as inferred in Pathway Tools. Considering the 121 C- and N-terminal positions in the protein [Several indices have been proposed to describe promiscuity ,52–55, t protein .π of a domain was defined as the product of the inverse versatility IV and the inverse abundance frequency IAF, as detailed in the Material and Methods section, Eqs IV describes the propensity of a domain to co-occur with other domains within a protein (and thus with other domains in a certain metabolic pathway). The promiscuity IAF accounts for how a given domain is specific to a given metabolic pathway in a certain strain. It is clear by definition that given a domain, both IV and IAF, and in consequence π, are strain-specific measures of promiscuity as IV and IAF depend on the number of proteins and pathways in a given strain, respectively. Surprisingly, for most of the domains, promiscuity is strongly strain-specific and, as shown in IV and IAF.The π rapidly decreases and converges towards smaller values (and thus higher promiscuity) when a sufficiently large number of organisms are considered, as can expected.To arrive at a measure of promiscuity that is strain-independent, we take a limiting approach, considering promiscuity as an ensemble property of a domain. We observed that for a large number of domains, the variation of Although domains may have low specificity, the number of possible biological functions that can be performed is limited as the number of possible partners is also limited. Decreasing in promiscuity, as a function of the number of strains, is a consequence of the fact that more domain configurations and functions are taken into account when more strains are added. The plateau reached by promiscuity curves indicates that no more information is gained by adding more strains and that possibly all biologically active domain configurations in streptococcus have been considered.π remains stable regardless of the number of organisms considered. de novo biosynthesis of purine. We call these domains “absolutely promiscuous domains”; by definition of Eqs π curves determines whether a domain is promiscuous or absolutely promiscuous. We explored behaviour of the promiscuity by fitting a straight line to the logarithm of π curves. The value of π when all species are considered is taken as the actual measure of the promiscuity of a given domain: the larger the π, the smaller the domain’s promiscuity.The shape of the A = 1/a of the slope a of the fitted line provides a measure of the absoluteness of the promiscuity of a given domain. Domains with smaller slope a, and thus larger A, tend to be absolutely promiscuous. To explore if a relationship exists between absolute promiscuity and promiscuity value, we plotted A versus π in the scatter plot given in π) and low absoluteness, indicating strain-specific promiscuity. For several domains, we observe low promiscuity (high π) and high absoluteness. For the remaining domains, we observe a large dynamic range of the index π, but small absoluteness.The inverse Streptococcus In the eukaryotes domain, promiscuity has been found to be a volatile, relatively fast-changing feature of eukaryotic proteins, with few domains remaining promiscuous throughout the evolution of eukaryotes [Streptococcus where a smaller fraction of domains remain promiscuous in all strains; this could be linked to the great extent of bacterial flexibility and to their capacity for rapidly adapting to changing environmental conditions by re-modulation of underground metabolism.This observation seems to contrast with the intuition that promiscuity should be a global property of a domain rather than a local one. The combination of domains with different structures and functions within the multi-domain proteins is a major mode of creation and modulation of molecular functionality ,55; the Comparing the metabolism of different species based only on the presence and absence of pathways provides only a limited view of their metabolic flexibility. Looking to metabolic pathways from a domain point of view, taking into account the promiscuity of the constituent domains, can provide insights on how and to what extent domains are reused in different metabolic pathways, thus providing an indication of metabolic flexibility and adaptability across different strains.To analyse the domain architecture of a metabolic pathway, we considered a metabolic pathway as an ensemble of domains obtained from collecting the entire domain architectures of the proteins involved in the metabolic reactions in the pathway. A given metabolic pathway is then described in terms of the promiscuity of the protein domains involved, as described by Eqs X and Y was calculated using the distance measure given in Because domain promiscuity is strain-dependent and domains show high variability between different strains, as previously discussed see Eqs , we therStreptococcus strain, we calculated D for each possible pair of functional metabolic pathways obtained in a squared domain-based (dis)similarity matrix. S. pneumonie. There are blocks of pathways with higher domain-based similarity, indicating distinct biological pathways that possess similar domain architecture. For instance, we observed that deoxyribonucleotide synthesis pathways and the glutamate metabolism pathways cluster together as they are functionally related. However, pathways that are functionally more distant also show high domain-based similarity, for instance the pathways for glycerol and ethanol degradation.For every Blocks of similar pathways may suggest the existence of sets of domain cores that act as building blocks for multiple metabolic pathways that are formed by incorporation and/or loss of domains, conferring to each species its metabolic individuality and flexibility.We observed that pathways with new functionality could be generated from existing pathways by the addition of one or more complete enzymes. However, the acquisition of an entirely new enzyme is a large evolutionary step for an organism. The question arises whether the remodulation of existing domain architectures within an organism would be sufficient for generating novel or alternative metabolic capabilities without the introduction of new enzymes. This would be consistent with the observation that functions of the individual domains in the multidomain proteins combine to produce their overall functions –9. PreliStreptococcus species, through the calculations of D for each possible pair of the 27 metabolic pathways that were found active in all of the 121 Streptococcus strains. Each strain results in a 27 × 27 symmetric matrix bearing the (dis)similarities between the 27 metabolic pathways described in terms of their domain promiscuity composition; these matrices can be visualised as the one given in We conclude by comparing the (dis)similarity of metabolic pathways within different What is of interest here is to investigate whether these dissimilarities among pathways are the same for all of the strains or are different between species. The INDSCAL modelling allows for a synthetic representation of the (dis)similarities among the core pathways as each matrix becomes a point in the INDSCAL space; the closer the point in space, the more similar the matrices are and thus, the more similar the domain architecture of the core domains.S. agalactiae (nine strains), S. pyogenes (19 strains) and S. suis (11 strains), and the other contains S. pneumoniae (26 strains), S. suis (six strains), S. thermophilus (six strains) and other minor strains. This clustering clearly indicates that the way the core metabolic pathways differ among each other is different, for instance, in S. pyogenes and in S. peneumoniae. In other words, it seems that for the strains analysed, the 27 core pathways appear in two major variants (corresponding to the two larger clusters) with smaller variants among the member of the same cluster. It should be remembered that in our approach, a vector whose elements are the absolute domain promiscuity represents each metabolic pathway. As domain promiscuity filters out strain-specificity, as previously discussed, this means that the core domains differ not only because of promiscuity, but also because of domain arrangements.When plotted on a three-dimensional space, we observe that the 121 species naturally cluster in two groups see  Panel A.The loading of the INDSCAL model can be inspected to highlight which metabolic pathways are more important for explaining the observed clustering, thus suggesting which metabolic pathways differentiate between the different strains. As shown in The higher loadings associated with these pathways indicate that the considered strains mostly differ in the relationships between these pathways and it is the differences between these pathways that cause the observed clustering between strains and species.S. suis species appears in both clusters, suggesting the existence of two different subspecies. On the basis of this, we performed a more in-depth analysis of the core metabolism of S. suis strains, which account for 40 functional pathways. When the same analysis is performed only on the S. suis strains, we observed that the serotype 2 strains cluster separately from strain with serotypes ½, 1, 3, 7, 9 and 14. It has been observed that serotype 2 S. suis strains are phylogenetically distinct from other serotypes [It is interesting to note that the erotypes , althougS. suis strains, we observed that several pathways are functional in S. suis 2, but not in the other serotypes and vice versa; in particular, we found that the UDP-α-D-glucuronate biosynthesis pathway (a precursor to capsular polysaccharides biosynthesis) is not present in S. suis 2 where an alternative UDP-N-acetylmuramoyl-pentapeptide biosynthesis I is found. The presence/absence of these two pathways discriminates between serotype 1 and other serotypes; nonetheless, we obtained separation of the serotype on the basis of the shared common pathways in which these pathways are not present. This indicates that different biological functions other than biosynthesis of capsular polysaccharides may be mirrored by serotype and that a comparison of different strains based on domain architecture may be more sensitive in highlighting biological and functional differences.Serotipisation is based on the presence of specific antigenic polysaccharides and the biosynthesis of capsular polysaccharides (cprs) requires an extremely complex pathway ,60. By cS. suis 2 is the most virulent form of the bacteria and is most often isolated in infected animals [S. suis 2 is markedly different from that of other serotypes. Exploring differences among the core metabolic pathways may provide hints for understanding the mechanism underlying the pathogenicity of S. suis 2, providing novel tools for diagnostic purposes and for epidemiological and transmission studies. animals ; it is a animals . Our anaStreptococcus strains were represented in terms of the domains participating in the metabolic reactions. This resulted in a numerical matrix representation of the metabolic capabilities of the considered strains, which allowed for in-depth statistical analysis.Bacterial species are able to adapt to different environments and conditions as a result of their metabolic flexibility. In this paper, we characterised proteins domains in terms of promiscuity, defined by adapting existing measures based on domain abundance and versatility. Subsequently, metabolic pathways that were inferred to be active in 121 Streptococcus suis and other strains. Our study indicates that comparative analysis of different organisms on the basis of their domain-based architecture can prove to be a complementary tool for comparative genomics.Through this approach, we could capture not only bacterial metabolic flexibility but also species diversity, indicating that it can be described in terms of reuse and sharing functional domains present in different proteins. We highlighted metabolic differences between species and even between strains within the same species, as is the case with the metabolic organisation of the pathogenic serotype 2 S1 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


From bacteria to eukarya, the specific recognition of the amino-acylated initiator tRNA by the universally conserved translational GTPase eIF5B/IF2 is one of the most central interactions in the process of translation initiation. However, the molecular details, particularly also in the context of ribosomal initiation complexes, are only partially understood.i, which, in contrast to the previous model, directly involve the methionylated 3′ CCA-end of the acceptor stem. These contacts are mediated by a conserved surface area, which is homologous to the surface areas mediating the interactions between IF2 and fMet-tRNAfMet as well as between domain II of EF-Tu and amino-acylated elongator tRNAs.A reinterpretation of the 6.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the eukaryal 80S initiation complex using the recently published crystal structure of eIF5B reveals that domain IV of eIF5B forms extensive interaction interfaces with the Met-tRNAThe reported observations provide novel direct structural insight into the specific recognition of the methionylated acceptor stem by eIF5B domain IV and demonstrate its universality among eIF5B/IF2 orthologs in the three domains of life. Finally, IF2⋅GTP catalyzes the joining of the 50S ribosomal subunit to form the elongation-competent ribosome . Doome . Ba. BafMet)ome . Ho. HofMet)i) to the small ribosomal subunit is carried out by the heterotrimeric a/eIF2, a specialized EF-Tu paralog that has no counterpart in bacteria . Th. Th20]. bacteria . Accordi joining ,19], and andi) totor stem .C. thermophilum, which shows a significantly higher degree of sequence similarity to the S. cerevisiae ortholog, allows a reinterpretation of the 6.6 Å resolution density. The resulting model demonstrates a direct contact between the methionylated CCA-end of the tRNA and a conserved surface area of domain IV that directly corresponds to the binding sites for the tRNA acceptor arm on domain IV of IF2 or domain II of EF-Tu , . In the 80S complex domain IV stabilizes the tRNA in a non-canonical P/I orientation  that accsolution , this insolution ,30]..i complei  The emergence of domain folds by polyphyletic evolution from self-assembling short peptide ancestors, whose remnants  still exist in extant proteins ; and ii)In light of these hypotheses, we suggest a possible common polyphyletic origin of both fold-related RNA binding interfaces discussed above. At the earliest stages of cellular evolution, when the fidelity of the primordial translation apparatus allowed the synthesis only of short peptides, nucleic acid-peptide interactions most likely played an essential role, particularly for the genetic machinery. In this context, it would be conceivable that the common ligand-binding interface in the ancient lineages of OB-fold proteins and translational GTPases has arisen as an ancient structural entity formed by individually synthesized peptides, associating with single-stranded nucleic acids as folding determinants, similar to metallo-peptides as precursors for metallo-proteins -39]; du; du39]; C. thermophilum to propose a new model for the interactions between eIF5B domain IV and the Met-tRNAi in the context of the ribosome  was performed using UCSF Chimera [i between bases G70 and A76 into the density next to domain IV was done in COOT [http://www.pymol.org).Rigid-body fitting of  Chimera . Despite in COOT . Figures in COOT  or PymolmtaIF5B crystal structure (PDB: 1G7R) is fitted to the cryo-EM density in the same way as the cteIF5B structure (with a CCC of 67%), supporting the newly proposed fit shown in Figure mtaIF5B domain IV of the cryo-EM based model (PDB: 4BYX) is fitted as presented in [α atoms) relative to the crystal structures of mteIF5B and cteIF5B, respectively, while the two crystal structures themselves differ only by an rmsd of 2.2 Å. Thus, the higher CCC for the cryo-EM-based model is most likely due to its distortion from the original rigid structure of mtaIF5B and is therefore not comparable to the CCC values obtained for our rigid-body fit.We would like to mention here that in our hands the isolated domain IV of the ure PDB: G7R is fiented in  with a Cure PDB: G7R is fiented in , which gMultiple sequence alignments were done using the iterative alignment program MUSCLE . StructuIF: Initiation factoreIF: Eukaryal initiation factoraIF: Archaeal initiation factorcryo-EM: Cryo-electron microscopypre-IC: Pre-initiation complexEMD: EM-DatabankEF-Tu: Elongation factor-Tuct: Chaetomium thermophilummt: Methanococcus thermoautotrophicumsc: Saccharomyces cerevisiaebs: Bacillus stearothermophilusPDB: Protein data bankCCC: Cross-correlation coefficientSCOP: Structural Classification of ProteinsOB-fold: Oligo-nucleotide/oligo-saccharide binding foldrmsd: Root mean square deviationPTC: Peptidyl-transferase centerASL: Anticodon stem loopThe authors declare that they have no competing interests.BK designed the study, analyzed structures and cryo-EM density, performed the structural modeling and sequence alignments, analyzed and interpreted the data and wrote the manuscript. RF analyzed the data and helped to draft the manuscript. Both authors read and approved the final manuscript.


The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer–dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.DOI:http://dx.doi.org/10.7554/eLife.06866.001 PTEN gene in cancer cells stop the PTEN protein working efficiently, or prevent PTEN production.Many cancers arise due to genetic mutations that allow cells to proliferate uncontrollably. Cell proliferation and many other cell processes can be regulated through a signaling pathway that involves an enzyme called PI3K. This ‘heterodimeric’ enzyme is made up of two protein subunits, one of which is called p85α and inhibits the other subunit of the enzyme (known as p110) to prevent uncontrolled cell proliferation. At the same time, p85α stabilizes p110 and allows the PI3K pathway to be briefly activated when appropriate. Many cancer cells contain mutations in the gene that encodes p85α that prevent the protein from inhibiting p110. This results in the activation of PI3K and promotes cancer formation. A protein called PTEN is a key inhibitor of the PI3K pathway. Common mutations to the Recent research has revealed that two molecules of p85α that are free from p110 can bind to each other to form a ‘homodimer’. Cheung et al. have now used biochemical, cell biological and computational methods to investigate the role of these p85α homodimers. This revealed that p85α homodimers stop PTEN being broken down by binding to it. As a consequence, there is enough PTEN in the cell to inhibit the PI3K pathway.By examining the mutations present in cancer patients, Cheung et al. next identified mutations that prevent the p85α protein from forming homodimers, or that prevent the homodimers from interacting with PTEN. PTEN therefore degrades and cannot inhibit the PI3K pathway, which allows the cells to proliferate. Methods that increase p85α homodimer formation or enhance the ability of p85α homodimers to bind to PTEN may therefore provide new approaches for developing cancer treatments. More generally, it appears that maintaining the correct balance between the amount of p85α in the form of p110-bound heterodimers and p110-free homodimers in a cell may be important for preventing diseases involving the PI3K pathway.DOI:http://dx.doi.org/10.7554/eLife.06866.002 PIK3R1, the gene coding for p85α, is the twelfth most frequently mutated gene across all cancers. Indeed, somatic mutations of the p85α:p110α interface disrupt the inhibitory action of p85α on bound p110α leading to PI3K pathway activation in cancers  pathway is a master regulator of many cellular processes. Activation of the class 1A PI3K is gated, in part, by the p85α regulatory subunit, which contains a Src homology 3 (SH3) domain, two proline-rich (PR) regions (PR1 and PR2) separated by a Rho-GAP/BCR-homology (BH) domain, and two Src homology 2 (SH2) domains  flanking an inter-SH2 (iSH2) domain. The interaction of p85α nSH2-iSH2-cSH2 fragment with the p110 catalytic subunit represses p110 activity allosterically and also stabilizes p110 against degradation, creating a pool of stable but quiescent p110 . Upon re cancers . HoweverPIK3R1 somatic mutation interferes with this process  . The difference in dimerization Kd between the N-terminal fragment and full-length p85α might indicate an additional entropic penalty arising upon full-length dimerization and/or weak intramolecular interactions occurring between the SH3-PR1-BH-PR2 and the nSH2-iSH2-cSH2 fragments in the monomer, which have to be displaced to allow dimerization.Previous analyses suggested that the region encompassing SH3-PR1-BH of p85α mediates p85α homodimerization and binding to PTEN . Howeverctively) . AUC andR/K]XXPXXP; R93PLP96VAP99)  p85α SH3 in a complex with a class I PXXP peptide that is similar to PR1  (The isolated SH3 domain does not form stable dimers in size-exclusion chromatography  and none96VAP99) . Indeed,of 24 μM . p85α SHof 17 μM , demonstof 17 μM . The lin μM  and (iid 16 μM) . We usedd 16 μM) .10.7554/We mutated either the key prolines or basic residues in the PR1 and PR2 regions proposed to bind p85α SH3 . These mRPLPPLPSL crystal structure (3I5R) (We next mutated p85α SH3 residues predicted to mediate binding to PR1 . The D21e (3I5R)  and our e (3I5R) , R18 weae (3I5R) .None of these mutants altered the interaction of p85α with p110α , consistThe p85α BH-PR2 fragment (residues 105–319) forms BH:BH domain dimers in two different crystal forms, suggesting that this dimer arrangement is not an artifact of a particular crystallization condition . The cry2 of the total solvent-accessible area of 9000 Å2, suggesting that the BH:BH interaction alone is insufficient for stable p85α homodimerization. Indeed, we measured a much lower affinity for p85α PR1-BH  than for constructs containing the SH3-PR1-BH region  regulates PTEN activity and stability, with the unphosphorylated counterpart (denoted hereafter as unphosphorylated PTEN) being more active . Consiste system . This ine system  or direce system . Importae system . The PIPted PTEN . While pted PTEN . Moreoveted PTEN , was incted PTEN . Of noteThe ubiquitin-proteasome pathway consists of a cascade of reactions with the substrate specificity being largely defined by E3 ubiquitin ligases. Therefore, we attempted to identify the PTEN E3 ligase that is regulated by the p85α homodimer. Consistent with previous studies , PTEN inTo validate this model, analytical gel filtration was performed to characterize the association and competition between p85α, p110α, PTEN, and WWP2. PTEN was recovered in fractions that were free of p110α  and bindNext, we determined whether complex formation is altered when p85α homodimerization is disrupted by the PR1+PR2mut. Intriguingly, while distribution of p110α was unchanged, there was a shift in the distribution of unphosphorylated PTEN to fractions lacking p85α , suggestPIK3R1 can contribute to tumorigenesis  and I177N  localizl cancer  and occul cancer . I177N al cancer   and likely others disrupt p85α homodimerization, including mutations at P99 in PR1, R162*, frame shifts at Q153, D168, H180, V181, and L182 , suggesting that some of these PTEN mutants may disrupt association with p85α therefore decreasing PTEN protein stability and activity.Based on our data, we speculate that the dynamic formation of p85α homodimers plays a role in the termination of signals initiated at the activation nidus by monomeric p85α-bound p110α. There are several possible scenarios in which the dynamics of p85α homodimer formation could be altered. For example, our data in this and previous studies  suggesteand L182 . FurtherIn addition to predicting mutations that disrupt homodimerization or PTEN interaction interfaces, the homodimerized p85α:PTEN model also offers possible mechanistic explanations for experimental observations. For example, the region outlined by I127/I133/E137 is close to in space to PR2 and both regions could thus together constitute a binding site for PTEN, possibly explaining the cooperativity between these residues in PTEN binding . The modOne key insight from our efforts to establish a speculative theoretical p85α:PTEN model was that our experimental constraints  cannot be satisfied by a structural model in which one PTEN phosphatase domain binds simultaneously to both BH domains of the p85α homodimer. Instead, our theoretical model proposes that the PTEN phosphatase domain binds to the BH domain of only one p85α protomer, opposite to the GTPase-binding site . To explPIK3R1 mutations could lead to PI3K pathway hyperactivation and thus contribute to diseases such as cancer. By decreasing PTEN expression and activity, these non-nSH2-iSH2-cSH2 PIK3R1 mutations may affect sensitivity to particular targeted therapeutics. Indeed, p110β rather than p110α has been proposed as the primary target in cells with PTEN protein loss  and Fyn SH3: PPRPLPVAPGSSKT (1A0N). This structure was refined using the Rosetta energy function as implemented in FlexPepDock (The complex between p85α SH3 and p85α PR1 peptide PxPepDock . ConstruE. coli BL21 cells as a 6His-fusion protein using the pET28b expression vector (Novagen). Protein production was induced by 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C. Cells were lysed using mild sonication, and proteins were purified to homogeneity by immobilized nickel affinity column using standard procedures. Eluted protein was dialyzed into 20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 5 mM DTT and applied to S75 (Pharmacia) size-exclusion chromatography. Fractions containing pure SH3 were pooled and concentrated.The p85α SH3 (residues 1–81) and PR1-BH domains (residues 79–301) were produced in coli BL21 cells as GST-fusion proteins using a pGex6P-2 expression vector . Protein expression was induced by 0.4 mM IPTG at 18°C overnight, and proteins were purified by GST affinity column. Eluted proteins were dialyzed into 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT. WT p85α was further purified using MonoQ 10/100 column and eluted by a linear NaCl gradient . Finally, p85α1-333 C147S and WT p85α were applied to a Superdex200 16/600  size-exclusion chromatography. Fractions containing pure proteins were pooled and concentrated.The p85α SH3-PR1-BH-PR2 fragment (residues 1–333) and WT p85α were produced in E. 647 according to the manufacture’s protocol . Labeled proteins were kept at 20 nM. The unlabeled protein was titrated starting at 50,000 nM and serially diluted in 1:1 ratio in reaction buffer . The measurements were performed at 40% LED and 20, 40, and 60% MST power on NanoTemper Monolith NT.115. Data were analyzed using NT Analysis Software .Proteins were fluorescently labeled with Alexa87KPRPPRPLPVAP into the measurement cell that contained p85α SH3 at a concentration of 100 μM. GST-ΔSH3 p85α and p85α SH3 titrations were carried out similarly, with SH3 in the syringe (400 μM) and ΔSH3 p85α in the cell. Ligand concentrations were 10× the cell concentration. Titrations were analyzed using Origin software.p85α SH3 and GST-ΔSH3 p85α were dialyzed in degassed ITC buffer . Titrations for p85α SH3:PR1 peptide interaction were carried out on a MicroCal ITC200 at 25°C by serially injecting 2 μl of peptide Pso20,w using SEDNTERP 1.09.The experiments were conducted on a Beckman Coulter XL-A analytical ultracentrifuge at 20°C using absorption optics. Samples were loaded at 17, 9, 5, 3, and 0.7 µM in a standard cell (400 μl). Data were acquired (120 scans) in intensity mode at 280 nm at 6-min intervals with a rotor speed of 48 krpm for p85α PR1-BH, 45 krpm for p85α SH3-PR1-BH-PR1, and 42 krpm for WT p85α. Data were analyzed in SEDFIT 14.3e  using thThe experiments were performed on a Beckman Optima XL-A analytical ultracentrifuge at 20°C. Samples were loaded at 1.0, 0.5, and 0.25 mg/ml into a 2-channel, 12-mm path-length cells (100 μl). Data were acquired at 11.8, 12.8, 13.8 krpm, as an average of 5 absorbance measurements at 280 nm using a radial spacing of 0.001 cm. Experiments were started from zero to the lowest rotor speed by taking scans at 2-hr intervals and testing for equilibrium by determining the differences between successive scans in SEDFIT. The rotor speed was then increased and the process repeated at each rotor speed studied and sedimentation equilibrium was achieved within 48 hr. Data for the individual proteins collected at different speeds and loading concentrations at 280 nm were sorted and assembled in SEDFIT and analyzed globally in terms of monomer–dimer–tetramer (monomer-m-mer-n-mer ) or monomer–dimer model in SEDPHAT 10.58e  with thePIK3R1 cDNA has been described (The KLE cell line was provided by Dr. Russell Broaddus (M.D. Anderson Cancer Center). The p85α knockout MEFs were obtained from Dr Lewis C. Cantley's lab . The escribed . Specifi308 or Ser473) and total p110α , HA , Myc and FLAG , ubiquitin  were used. For IP, cell lysates (1 mg) were immunoprecipitated with antibodies against HA or Flag (1 μg) or PTEN (1:500) overnight at 4°C. The immune complexes were collected by incubation with protein A/G agarose (Santa Cruz) for 4 hr before being resolved by SDS-PAGE.Whole cell lysates (25 µg) extracted with radioimmunoprecipitation assay buffer (RIPA) lysis buffer were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Primary antibodies specific to PTEN, p110α, total Akt, phospho-Akt  clarified by ultracentrifugation  were applied to Superose 6, 10/300 GL column  run at 4°C in binding buffer (0.1% NP-40) on a BioLogic HR system . Elution was performed at 0.1 ml/min, and 0.5-ml fractions were collected. Fractions were analyzed by Western blotting (WB) or pooled for IP with anti-p85α (1:50)  or PTEN (1:500) antibody.Cells were lysed in a hypotonic lysis buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgClPIK3R1 or mutants were cultured in medium without IL-3 for 4 weeks. Cell viability was evaluated using PrestoBlue (Invitrogen) for mitochondrial dehydrogenase activity.The assay was described previously . In brie3 substrate was added to initiate the reaction. Reaction mixture with PIP3 but not lysate was used for background correction. The reaction was stopped after 4-hr incubation at 37°C and the supernatant was separated from the beads for activity detection. The beads were used for WB to quantify immunoprecipitated PTEN. The activity was normalized to immunoprecipitated PTEN level in each sample. To assess the activity of recombinant PTEN, 1 μg of PTEN was incubated with 1 μg of recombinant p85α in buffer containing 50 mM Tris–HCl pH 8.0, 50 mM NaCl, and 10 mM MgCl2 (PTEN reaction buffer without DTT) at room temperature for 1 hr before being subjected to the reaction.In vitro PTEN lipid phosphatase activity was determined using malachite green phosphatase assay kit . Briefly, protein lysates (1 mg) were subjected to IP of endogenous PTEN using anti-PTEN or anti-p85α antibody and the bead complex was resuspended in PTEN reaction buffer before PIPg for 10 min, the phospholipid pellets were dissolved in SDS sample buffer. The proteins in the supernatants corresponding to the lipid-unbound population were precipitated with 20% trichloroacetic acid and the precipitates were dissolved in SDS sample buffer. Equal amounts of samples from the supernatants and pellets were analyzed by SDS-PAGE and Coomassie Brilliant Blue R-250 staining.The assay was performed in reference to previous studies . FormatiAll experiments were independently repeated at least twice, and data are presented as mean values ± SD. The significance of differences was analyzed by a Student’s t test. Significance was accepted at the 0.05 level of probability (p < 0.05). review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Regulation of the PI3K Pathway through a p85α Monomer-Homodimer Equilibrium” for consideration at The Reviewing Editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.This is a carefully performed study that combines biochemical analysis and structural modeling to gain insight into the regulation of PTEN. A major conclusion of the study is the p85 homodimers compete for binding PTEN with the WWP2 E3 ligase. The conclusions of the study are, in general, consistent with the analytical data presented. However, in some instances the conclusions are over-stated and in other instances alternative interpretations of the data are not considered.1) 2) The competition model is compelling, but it appears to be based on over-expression/transfection assays. Studies of endogenous proteins/loss-of-function analysis are needed to provide more convincing support for the authors' conclusions. For example, does knock-down of WWP2 increase the co-immunoprecipitation of p85 with PTEN (and the reverse experiment).3) The interpretation of the transfection assays is limited to the deduction of direct binary interactions between PTEN with p85 homodimers and/or WWP2. Possible roles for signaling changes in the cells transfected with these reagents are not considered. For example, do changes in the PI3K pathway contribute to the changes in the PTEN interactions described? Feed-back or feed-forward signaling mechanisms may play a contributing role.4) Are the biochemical interactions described for p85 in this study specific to p85α or does p85β similarly homodimerize and associate with PTEN through the indicated regions? The potential for redundancy in these mechanisms becomes important when one considers the influence of cancer-associated PIK3R1 mutations.5) The structural model was constructed through a complicated procedure described in an eight page supplement, which describes some straightforward approaches  and others that are more tenuous (e.g. BUNCH with synthetic SAXS data). The resulting composite model depends on multiple assumptions, with some supporting biochemical/structural constraints, but is nevertheless speculative as the authors acknowledge. A triple mutation on a predicted PTEN-binding surface of the BH domain suggests that a least one of the mutated residues is located in the interface. However, there are no experiments that assess the structure in solution . It is difficult to assess the overall likelihood/uniqueness of the model or whether the constraints are sufficient to exclude alternative possibilities. In the absence of direct structural data for the relevant complex, the authors should be more circumspect with respect to the presentation of the speculative model and its implications, including discussion of potential caveats and possible alternative interpretations.Kd for dimerization of the SH3-PR1-BH-PR2 fragment of p85α is 10 fold lower than for the full length protein, which is a non-trivial difference. How do the authors explain this result?6) The  This is a carefully performed study that combines biochemical analysis and structural modeling to gain insight into the regulation of PTEN. A major conclusion of the study is the p85 homodimers compete for binding PTEN with the WWP2 E3 ligase. The conclusions of the study are, in general, consistent with the analytical data presented. However, in some instances the conclusions are over-stated and in other instances alternative interpretations of the data are not considered.1). In the subsection headed “Disruption of p85α homodimerization is associated with decreased stability of PTEN through ubiquitination”, it is stated that WT p85 decreases and mutant p85 increases ubiquitination. This is true in a relative sense, but it appears that the extent of ubiquitination in each case is decreased compared with the LacZ control. These data should be presented more clearly.The extent of PTEN ubiquitination in LacZ-transfected cells was higher than in p85α mutant-transfected cells because the amount of PTEN-bound p85α homodimer, which inhibits PTEN ubiquitination, was higher in mutant-transfected cells. The mutants, although they display decreased interaction with PTEN compared with wild-type p85α, were not completely defective in PTEN binding. We have now clarified this in the text (in the first paragraph of the subsection headed “Disruption of p85α homodimerization is associated with decreased stability of PTEN through ubiquitination”).2) The competition model is compelling, but it appears to be based on over-expression/transfection assays. Studies of endogenous proteins/loss-of-function analysis are needed to provide more convincing support for the authors' conclusions. For example, does knock-down of WWP2 increase the co-immunoprecipitation of p85 with PTEN (and the reverse experiment).In response to the reviewers’ comments, we have performed loss-of function studies using siRNAs that efficiently target WWP-2 or p85α in HEC1A which is an endometrial cancer cell line expressing high levels of WWP-2 and p85α. The interaction between PTEN and p85α, or between PTEN and WWP-2 was increased after knockdown of WWP-2 or p85α respectively. The data are now presented in 3) The interpretation of the transfection assays is limited to the deduction of direct binary interactions between PTEN with p85 homodimers and/or WWP2. Possible roles for signaling changes in the cells transfected with these reagents are not considered. For example, do changes in the PI3K pathway contribute to the changes in the PTEN interactions described? Feed-back or feed-forward signaling mechanisms may play a contributing role.We agree with the reviewers that it would be interesting to examine whether the PTEN/p85 α/WWP2 interactions are subjected to changes in PI3K pathway signaling, especially through altering feedback and feedforward mechanisms. Negative feedback mediated by S6K  and posiTo investigate whether these regulatory mechanisms could affect the interaction of PTEN with p85α or WWP2 when these molecules are altered by transfection, we suppressed S6K or mTORC2 signaling using S6K or Rictor siRNA, respectively, and rapamycin was used to inhibit mTORC1 activity in the cell line used in this study (KLE). In addition, we also targeted AKT activity using the specific AKT inhibitor MK2206. S6K siRNA increased AKT phosphorylation implicating the existence of negative feedback in KLE cells. In contrast to the suggested role in mediating a positive feedback at least in some cell lines, Rictor siRNA did not decrease phosphorylated AKT in the KLE cell line, although the siRNA decreased Rictor expression by 80%. Importantly, both S6K and Rictor siRNA had no effect on PTEN interactions with p85α or WWP2. Rapamycin and MK2206, which efficiently inhibited mTOR and AKT phosphorylation, respectively, also had no effect on these PTEN interactions with p85α or WWP. Thus, inhibition of these feedback and feedforward loops, and inhibition of different nodes of the PI3K pathway, does not affect the degree of PTEN association with p85α or WWP2, suggesting that the PTEN interactions with p85α or WWP2 are unlikely to be explained by mutant-triggered changes in PI3K downstream signaling or regulatory loops. These data and discussion are now added in 4) Are the biochemical interactions described for p85 in this study specific to p85α or does p85β similarly homodimerize and associate with PTEN through the indicated regions? The potential for redundancy in these mechanisms becomes important when one considers the influence of cancer-associated PIK3R1 mutations.The extent of functional redundancy between p85α and p85β is an interesting area of investigation. Harpur et al. have reported the homodimer formation of recombinant p85β proteins using size exclusion chromatography . To furtThe nSH2-iSH2-cSH2 fragment that binds p110 is 80% identical in sequence between p85α and p85β. In contrast, p85α and p85β share only 30% protein sequence identity between their BH domains that are critical for PTEN binding. Importantly, the BH region that mediates dimerization in p85α is not conserved in p85β, neither in sequence nor in structure . Accordingly, the BH domain dimer centered on M176 present in the p85α crystal is not present in the crystal lattice of p85β BH see . Instead5) The structural model was constructed through a complicated procedure described in an eight page supplement, which describes some straightforward approaches  and others that are more tenuous (e.g. BUNCH with synthetic SAXS data). The resulting composite model depends on multiple assumptions, with some supporting biochemical/structural constraints, but is nevertheless speculative as the authors acknowledge. A triple mutation on a predicted PTEN-binding surface of the BH domain suggests that a least one of the mutated residues is located in the interface. However, there are no experiments that assess the structure in solution . It is difficult to assess the overall likelihood/uniqueness of the model or whether the constraints are sufficient to exclude alternative possibilities. In the absence of direct structural data for the relevant complex, the authors should be more circumspect with respect to the presentation of the speculative model and its implications, including discussion of potential caveats and possible alternative interpretations.Importantly, the hypothetical model presented is not used as a proof for our conclusions or indeed as a stand-alone model to explain the interactions between p85α monomers or between p85α and PTEN or WWP2, but to rather to guide and inform the experiments to be performed to elucidate the functional role of particular residues in p85α. We had emphasized this in the text and now clarify this further . To be more circumspect with respect to the presentation of the speculative model, we have limited its presentation in the manuscript from three to only one figure  The Kdfor dimerization of the SH3-PR1-BH-PR2 fragment of p85α is 10 fold lower than for the full length protein, which is a non-trivial difference. How do the authors explain this result?Kd than the full-length protein. These differences are reproducible and significant. In AUC, these differences are also robust with respect to the model used for fitting, and the type of data acquisition (sedimentation equilibrium or velocity). Determination of the molecular basis of this observation would require complete experimental determination of structures of dimeric and monomeric p85 α (full-length and fragments thereof). Such structural analysis is non-trivial because of the size and flexibility of this multi-domain protein, and hence is beyond the scope of our manuscript.Indeed, the shorter N-terminal fragment consistently displayed a ten-fold lower dimerization Kd between the N-terminal fragment and full-length p85α might indicate an additional entropic penalty arising upon full-length dimerization…”).Nonetheless, we speculate that the mildly inhibitory nature of the C-terminal nSH2-iSH2-cSH2 fragment arises from additional entropic penalty upon full-length dimerization, and/or from weak intramolecular interactions occurring between the SH3-BH fragment and the nSH2 -iSH2-cSH2 fragment in the monomer, which have to be displaced upon dimerization. This possibility has now been included in the Results section (“The difference in dimerization


MID1 catalyzes the ubiquitination of the protein alpha4 and the catalytic subunit of protein phosphatase 2A. Mutations within the MID1 Bbox1 domain are associated with X-linked Opitz G syndrome (XLOS). Our functional assays have shown that mutations of Ala130 to Val or Thr, Cys142 to Ser and Cys145 to Thr completely disrupt the polyubiquitination of alpha4. Using NMR spectroscopy, we characterize the effect of these mutations on the tertiary structure of the Bbox1 domain by itself and in tandem with the Bbox2 domain. The mutation of either Cys142 or Cys145, each of which is involved in coordinating one of the two zinc ions, results in the collapse of signal dispersion in the HSQC spectrum of the Bbox1 domain indicating that the mutant protein structure is unfolded. Each mutation caused the coordination of both zinc ions, which are ∼13 Å apart, to be lost. Although Ala130 is not involved in the coordination of a zinc ion, the Ala130Thr mutant Bbox1 domain yields a poorly dispersed HSQC spectrum similar to those of the Cys142Ser and Cys145Thr mutants. Interestingly, neither cysteine mutation affects the structure of the adjacent Bbox2 domain when the two Bbox domains are engineered in their native tandem Bbox1-Bbox2 protein construct. Dynamic light scattering measurements suggest that the mutant Bbox1 domain has an increased propensity to form aggregates compared to the wild type Bbox1 domain. These studies provide insight into the mechanism by which mutations observed in XLOS affect the structure and function of the MID1 Bbox1 domain. We recently demonstrated that MID1 catalyzes the polyubiquitination of the catalytic subunit of protein phosphatase 2A (PP2Ac) (Du (2)-C-x(7–10)C-x(2)-C-x(4–5)-C-x(2)-C(H)-x(3–6)-H-x(2–8)-H 5(C/H)H2][C. The second Bbox domain  is approximately 40-45 amino acids with the consensus sequence: C-x2-4-C/H-x7-10-C-x7-C-x2C-x3-6-H-x2-8-H [3H2C(C/H)C/DC]. The tertiary structures of both types of Bbox domains reveal that each coordinates two zinc ions in a cross-brace manner and adopts the ββα-RING fold typically observed in RING E3 ligases MID1 has two consecutive zinc-binding Bbox domains that follow the N-terminal RING domain Thus, MID1 is a novel E3 ligase with three consecutive RING E3 ligase domains. The RING domain exhibits greater E3 ligase activity compared with either Bbox domain et al., PLoS One, manuscript in review).Alpha4 binds MID1 via the Bbox1 domain and this interaction is important for the MID1-mediated polyubiquitination of alpha4. Supporting this observation, a Leu146Gln mutation within the Bbox1 domain disrupts the interaction and the polyubiquitination of alpha4 et al., PLoS One, manuscript in review) suggesting that these mutations prevent the interaction of MID1 with alpha4.Mutations of MID1 are associated with X-linked Opitz G Syndrome (XLOS), which is characterized by cleft lip/palate, hypertelorism and hyperspadias 1H-15N NMR spectroscopy to characterize the effects of these mutations on the tertiary structural properties of the MID1 Bbox1 domain. The data reveal that although the mutations involve residues  closely associated in the structure with only one of the two bound zinc ions, the coordination of both zinc atoms is lost, resulting in protein unfolding and aggregation.Here we used two-dimensional (2D) 6-tag. The Bbox1-Bbox2 construct included residues from the linker region that follow the RING domain (residues 71–110) and is referred to as LB1B2. The Ala130Thr, Ala130Ser, Cys142Ser and Cys145Thr point mutations within the Bbox1 domain were performed using a standard PCR mutagenesis protocol. The sequences of the DNA insert and mutations were confirmed by DNA sequencing before the plasmids were transformed into E. coli BL21(DE3) cells.The DNA encoding the MID1 Bbox1 domain (residues 110–164) and the Bbox1-Bbox2 were cloned into the pET151/D-TOPO vector containing the sequence of a C-terminal His15N-labeled protein for NMR studies, cells were grown in M9 minimal media supplemented with trace metals, vitamins, glucose and 15N-NH4Cl. Cells were grown at 37°C to an OD600 of ∼0.6 and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 4 hr at 37°C. Harvested cells were stored at −80°C and then re-suspended in 50 mM Tris (pH 7.7), 200 mM NaCl, 10 mM β-mercaptoethanol, 1 mM ZnCl2 and 2% sarkosyl, followed by lysis with sonication g at 4°C for 30 min. The proteins were purified using a standard Ni2+-NTA affinity chromatography approach. The C-terminal His6-tag was not removed because of a lack of a protease cleavage site between the protein and the tag.To obtain 1H-15N heteronuclear single quantum coherence (HSQC) spectra were acquired at 25°C with 0.3 to 0.5 mM 15N-labeled wild type and mutant Bbox1 and LB1B2 proteins using a Varian DD2 600 MHz spectrometer equipped with a 5 mm triple resonance  probe with z-axis gradient. The NMR data were processed with nmrPipe Two-dimensional g) derived from the corresponding tertiary protein structures using CRYSOL Dynamic light scattering (DLS) data were acquired with a Wyatt Technology DynaPro Nanostar instrument. Three measurements, each with ten acquisitions, were acquired for each protein sample in 100 µL volume. The concentration of the wild type and mutant Bbox1 and Bbox1-Bbox2 proteins was in the range 50–100 nM. DLS data were also acquired for ubiquitin (2 mM) and lysozyme (6.8 mM) to serve as controls. The DLS measurements were benchmarked to predictions of the radius of gyration . N and 15N atoms were observed over the range of 7.5–10.1 ppm and 105–129 ppm, respectively. In contrast, the cross peaks for the Cys142Ser Bbox1 domain were not well dispersed, with the HN and 15N signals resonating between 7.9–8.5 ppm and 118–127 ppm, respectively  ethylenediaminetetraacetic (EDTA). EDTA is a potent divalent metal chelator. If the Cys142Ser mutant Bbox1 domain retains the ability to coordinate any zinc ions, then EDTA is expected to compete for metal binding and cause additional chemical shift changes in the spectrum. As shown in To confirm that the spectrum of the Cys142Ser Bbox1 domain was consistent with the loss of both zinc ions, and to show that the addition of EDTA to the Cys142Ser mutant  would haWe studied the effect of the Cys145Thr mutation on the structure of tandem Bbox1 and Bbox2 domains using the LB1B2 construct (MID1 residues 71–214). Residue Cys145 is coordinated to the same zinc ion as Cys142 and we anticipated that the Cys145Thr mutation would have a similar destabilizing effect on the structure of the Bbox1 domain. Therefore, we used the Cys145Thr mutation to investigate how an unstructured Bbox1 domain would affect the structure of the adjacent Bbox2 domain.1H-15N-HSQC spectrum of the Cys145Thr mutant LB1B2 protein is shown superimposed with that of the corresponding wild type protein in 15NH signals for the Cys145Thr mutant LB1B2 were collapsed to the center of the spectrum, with poor signal dispersion in both the HN and 15N dimensions. This observation is consistent with the presence of a substantial component of a disordered protein structure, assumed to correspond to the destabilized Bbox1 domain. The additional number of poorly dispersed cross peaks in the center of The Residue Ala130 is highly conserved in Bbox1 domain sequences, particularly in the C-I subclass of TRIM proteins consisting of COS, FNIII and B30.2 C-terminal domains 15N–H cross peaks with very little chemical shift dispersion in either dimension (The HSQC spectrum of the Ala130Thr Bbox1 domain showed imension . Compariimension . With thN dimension (7.4–8.7 ppm) compared to that of the wild type Bbox1 domain. However compared to the spectrum of the Ala130Thr Bbox1 domain, more signals were observed and the peaks were slightly more spread out  construct harboring the Ala130Ser substitution to catalyze the ubiquitination of alpha4. As shown in et al., PLoS One, manuscript in review).Because the HWe investigated the structural effects of the Ala130Thr mutation on the adjacent Bbox2 domain within the LB1B2 protein construct. According to the tertiary structure of the Bbox1-Bbox2 domains in tandem, residue Ala130 is located relatively close to the interface of the two domains  [39]. ThWe compared the HSQC spectra of the wild type and Ala130Thr LB1B2 proteins . The speNatural mutations of MID1 are shown to form cytoplasmic clumps in cells, instead of its normal association with microtubules g) derived from the DLS measurements, we utilized the program CRYSOL g based on the known tertiary structures of these proteins . Furthermore, the data indicated that 25% of the population of the Ala130Thr Bbox1 protein formed very large soluble aggregates (Rg >300 Å). Similarly, the DLS data suggested that the Cys142Ser Bbox1, Ala130Thr LB1B2, and Cys145Thr LB1B2 proteins could be in equilibrium between dimeric and larger soluble aggregate states. Approximately 70% of the population of the Ala130Ser Bbox1 mutant protein had a Rg of ∼18±7 Å, consistent with a monomer-dimer equilibrium.We acquired DLS data of the individual wild type and mutant Bbox1 domain proteins (MW 6 kDa) alone and in tandem with the Bbox2 domain . As a benchmark, we acquired DLS data for ubiquitin (8 kDa) and lysozyme (14 kDa). To validate the radius of gyration  indicating that the Bbox2 domain can compensate for the loss of function of the Bbox1 domain.When expressed in the context of a Bbox1-Bbox2 tandem construct (MID1 (residues 71–164), all three mutations affected only the structure of the Bbox1 domain. Even though Ala130 is located near the interface, its mutation contributes little to the Bbox2 domain stability since the structure of the Bbox2 domain remains unperturbed. As such, it is possible that full-length MID1 harboring Bbox1 mutations could still maintain some functionality that is independent of the Bbox1 domain. For example, the RING-Bbox1-Bbox2 (RB1B2) constructs harboring the various Bbox1 domain mutations were capable of catalyzing the polyubiquitination of PP2Ac (Du In summary, we used NMR spectroscopy to demonstrate that a number of XLOS-observed mutations within the Bbox1 domain cause the structure of the Bbox1 domain to unfold. The mutations also cause the isolated Bbox1 and Bbox1-Bbox2 proteins to form soluble aggregates. It is therefore conceivable that within the full-length MID1 these mutations might contribute to the formation of cytoplasmic clumps observed in XLOS-derived fibroblasts


Toxin production by pathogenic microorganisms likely serves to (i) protect against phagocytosis by predatory cells, (ii) aid in penetrating tissue barriers, (iii) promote nutrient release, or (iv) alter cellular architecture and metabolism in ways that facilitate the establishment of a niche for colonization and replication. Useful enzymatic targets for some bacterial toxins may be similar between prokaryotic and eukaryotic environments, necessitating a need for eukaryotic-specific cofactors to regulate toxin activity. Host cell—derived factors may impart localization properties to an effector, induce folding events, provide a platform for the inhibition of cellular processes or support greater substrate promiscuity . The aimP. aeruginosa. Monoubiquitin, ubiquitin polymers, and ubiquitylated proteins are capable of activating ExoU [P. asymbiotica, B. thailandensis, and P. fluorescens [Many bacterial toxins contain catalytic domains with homology to plant patatins, which are lipid acyl hydrolases found in potato tubers. Cofactor activation of phospholipase activity is best characterized for ExoU, a lipid membrane—hydrolyzing protein encoded by the opportunistic pathogen ing ExoU . Bioinfoing ExoU . Functioorescens .R. typhus encodes ExoU homologs, Pat1 and Pat2 [R. prowazekii, the etiologic agent of typhus, displays enhanced enzymatic activity in the presence of bSOD1 , whiciquitin, ). Ricketar entry , phagosoar entry .L. pneumophila displays a Rab5-dependent PLA1 activity [Salmonella, is activated by GTP-RhoA [Y. enterocolitica homolog to SseJ, YspM, may have a similar function [VipD lipase from activity  that tarGTP-RhoA  likely fGTP-RhoA . The Y. function .P. aeruginosa was one of the first identified bacterial ADP-ribosyltransferases requiring a cofactor for activation [A. hydrophila [V. parahaemolyticus [Most cofactor-stimulated transferases catalyze the transfer of an ADP-ribose moiety from nicotinamide adenine dinucleotide to a target protein. Exoenzyme S from tivation . Memberstivation . Homologdrophila  and V. polyticus .P. aeruginosa, ExoT, is similar (76% identity) to ExoS and requires 14-3-3 proteins as cofactors, but has limited target recognition and is not overtly cytotoxic [A. hydrophila and A. salmonicida encode enzymes similar to ExoT/S termed AexT [Another enzyme from ytotoxic . CrkI anytotoxic . ADP-ribmed AexT .Yersinia that inhibits immune responses through acetylation of several residues in the kinases involved in the MAPK and NFκB pathways [P. syringae secretes an IP6-activated acetyltransferase, HopZ1, which interferes with plant cell cytoskeletal networks and downstream cell wall—mediated immune responses by acetylating microtubules [S. typhimurium AvrA suggests that stimulation of acetyltransferase activity in this family of enzymes likely involves allosteric conformational changes [V. cholerae RTX toxin [Clostridium difficile toxin A [Bacterial effectors with acetyltransferase activity include YopJ, a type III secreted enzyme from pathways . Acetyltpathways ). P. syrotubules . The bin changes . StructuTX toxin  and Clos toxin A  suggest V. cholerae, LT-I and LT-II from E. coli, are the most studied enzymes of this family to date; each are highly homologous in sequence and structure. Cholera toxin A1 subunit was shown to fold on host cell lipid rafts [s), locking it into the GTP-bound form. This stimulates adenylyl cyclase activity and generates supraphysiological amounts of cAMP, deregulating the chloride balance in the intestinal lumen to cause diarrhea.ADP-ribosylation factors (ARFs) have been identified as stimulators of type I and type II heat labile toxin (LT) activity from enteric bacteria. Cholera toxin from id rafts  before iid rafts . ActivatB. pertussis. These enzymes contain structurally similar components but differ in affinity and binding mechanism for their eukaryotic activator, calmodulin (CaM). Edema factor has three domains: a protective antigen-binding domain, the adenylyl cyclase core domain, and an alpha-helical domain. The helical domain interacts with the core domain to maintain an unstructured activation loop. The N-terminal domain of CaM interacts with the EF helical domain, causing a conformational switch between the helical and core domains [Cofactor-stimulated nucleotide cyclases include the adenylyl cyclases edema factor (EF) of anthrax toxin and CyaA of  domains . The C-t domains .In contrast to soluble EF, CyaA localizes to the cytosolic interface at the plasma membrane. The CaM binding domain involves completely different sequences with an affinity that is roughly 100-fold stronger as compared to EF. N- and C-terminal domains of CaM participate in binding, stabilization, and activation of CyaA. The C-terminal domain stabilizes a catalytic loop, and the N-terminal domain is postulated to bind a β-hairpin motif of CyaA to promote substrate (ATP) binding within the catalytic chamber .P. aeruginosa [Exoenzyme Y is a type III-secreted, cofactor-stimulated adenylyl cyclase from ruginosa . Functioruginosa . The cofruginosa . Sensitiruginosa ,33. The ruginosa .Yersinia species inject actin-stimulated effector kinases into host cells [st cells . YopO orst cells .Shigella species, but also present in strains of Yersinia and enterohemorrhagic E. coli, is OspG [Another kinase characterized from  is OspG –39. OspG is OspG . Ubiquit is OspG  and UbcH is OspG . At leasV. parahaemolyticus has recently been shown to secrete an IP6-activated enzyme, VPA1380, with homology to the cysteine protease domains of multifunctional-autoprocessing RTX (MARTX) domains from Vibrio and large clostridial toxins A and B [P. syringae secretes a protease, AvrRpt2, which is homologous to the staphopain cysteine proteases that are activated through interactions with cyclophilins [ A and B . The mecophilins . AvrRpt2ophilins . Analysiophilins .Numerous secreted bacterial enzymes interact with host cell factors to ensure enzymatic activation in the correct environment, and in some cases, trafficking to a specific location within the host. Common properties of the activators are that they are ubiquitously distributed throughout the eukaryotic kingdom, are present in high concentration, and play roles in cell signaling. The prevailing theme of a high cellular concentration may facilitate the required conditions for toxins to establish a cross kingdom binding partner as they evolved . In this


Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a group of immunoglobulin-related vertebrate glycoproteins. Several family members, including CEACAM1, CEA, and CEACAM6, are found on epithelial tissues throughout the human body. As they modulate diverse cellular functions, their signaling capacity is in the focus of current research. In this review we will summarize the knowledge about common signaling processes initiated by epithelial CEACAMs and suggest a model of signal transduction by CEACAM family members lacking significant cytoplasmic domains. As pathogenic and non-pathogenic bacteria exploit these receptors during mucosal colonization, we try to highlight the connection between CEACAMs, microbes, and cellular responses. Special emphasis in this context is placed on the functional interplay between CEACAMs and integrins that influences matrix adhesion of epithelial cells. The cooperation between these two receptor families provides an intriguing example of the fine tuning of cellular responses and their manipulation by specialized microorganisms. CEACAMsme 19q13 . Whereasme 19q13 . Some fame 19q13 ,8. Howevme 19q13 ,9. Givenme 19q13 , we willSince the discovery of carcinoembryonic antigen (CEA) some 50 years ago , and thehttp://www.carcinoembryonic-antigen.de/index.html; [V-like domain, followed by three  or two  IgC2-like domains, respectively. Similarly, in other epithelial CEACAMs, such as CEA or CEACAM6, up to six extracellular IgC2-like domains follow the amino-terminal IgV-like domain . Invex.html; ). The maTrans-oligomerization resulting from homophilic interactions between the amino-terminal IgV-like domains of CEACAMs on neighbouring epithelial cells is the basis of CEACAM-mediated cell-cell adhesion [trans-oligomerization is further supported by the presence of IgC2-like domains [trans-interactions [V-like amino-terminal domain for trans-oligomerisation, but also pointed to additional cis-interactions in the extracellular part of CEACAM1 [cis-interactions between either IgV-like domains or IgC2-like domains of parallel CEACAM1 molecules in the same membrane plane [trans- or cis-interactions between their extracellular domains. At least in the case of CEACAM1, these different oligomerization states clearly have an influence on its signaling function [cis-interactions, presumably depending on the lipid context.adhesion -36. Howe domains ,37. In aractions ,38. More CEACAM1 . Indeed,ne plane . As a cofunction . In one FGKTGSSGPLQ), respectively, interact with cytoskeletal components and T457 (-HFGKTGSSGPLQ) is phosphorylated [As transmembrane signaling requires a connection to the cytosol, the transmembrane domain containing CEACAM1 has been the focus of a multitude of studies ,13. Indeorylated . Furtherorylated . In thisorylated .V-like amino-terminal domain, which is instrumental in CEACAM1 for homophilic interactions between CEACAM1 on neighbouring cells. Together, these recent insights point to functional commonalities between epithelial CEACAM family members, which show striking sequence divergence in their amino-terminal IgV-like domain or their cytoplasmic sequences.A similar contribution of CEACAM1 to morphogenesis has now been reported in 3D cultures of prostate cells . The priV-like domain, such as the transmembrane or additional extracellular domains. Indeed, recent experiments employing either carcinoma cell lines or using bacterial pathogens as CEACAM ligands have pointed into this unexpected direction.One important implication arising from these results is the realization that signaling by epithelial CEACAMs could involve parts of these receptors other than the cytoplasmic domain or the amino-terminal Igcis-dimer formation of CEACAM1. Recent biochemical approaches based on chemical crosslinking support the idea that CEACAM1 oligomerizes laterally via the transmembrane domain to sustain downstream function [A long standing observation in the field is the reduced expression of CEACAM1 that accompanies transformation of epithelial cells from different tissues , includifunction . TogetheEscherichia coli strains, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis, have been found to bind to CEACAM1 or other epithelial CEACAMs such as CEA and CEACAM6 [Further insight into CEACAM signaling connections has been gained by the use of bacterial pathogens as selective and multivalent stimuli of these receptors. Over the last two decades, diverse CEACAM-binding pathogens including pathogenic  CEACAM6 -56. In a CEACAM6 ). Indeed CEACAM6 . In addi CEACAM6 ,58,59. I CEACAM6 ,61. Due  CEACAM6 . Importa CEACAM6 . This is CEACAM6 . Therefo CEACAM6 . In this CEACAM6 . Transme CEACAM6 ,67. In cC2-like domains of CEACAM1 [C2-like domains resulted in lower numbers of endocytosed bacteria in comparison to wildtype CEACAM1 despite a similar binding of the microorganisms to the truncated receptor. It is interesting to note, that inhibition of PI3K by wortmannin did not interfere with the re-location of CEACAM1 to membrane microdomains, suggesting that PI3K signaling is downstream of receptor oligomerization and membrane microdomain association of the receptor. A plausible explanation would be that the IgC2 domains of CEACAM1 connect bacteria-bound CEACAM1, presumably via the extracellular part of a membrane-microdomain located receptor, with PI3K signaling inside cells  play an important role ,75. Ther CEACAM1 . AccordiV-like domain [V-like domain compared to the IgC2-like domains has always been interpreted as a sign of positive selection for sequence variation in the amino-terminal domain. However, together with the loss of function upon deletion of the IgC2 domains, the relative conservation of the IgC2 domains of epithelial CEACAMs may reflect conserved functions and therefore evolutionary constraints on this region. Importantly, whereas all isoforms of CEACAM1, CEA, and CEACAM6 encompass at least one IgC2-like extracellular domain, CEACAM3 lacks such an extracellular domain. The absence of an IgC2-like extracellular domain in CEACAM3 correlates well with the mechanistically distinct endocytosis mediated by CEACAM3 in comparison to epithelial CEACAMs. Altogether, it is very tempting to speculate that engagement of epithelial CEACAMs will promote the association of their extracelluar IgC2 domain(s) with not yet identified co-receptor(s), which in turn transmit the PI3K activating signal into the cell (Figure It is interesting to note that the IgC2 domains of CEACAM1 orthologs from human, cattle, mouse and rat show a higher degree of sequence conservation than the amino-terminal Ige domain ,78. The Moraxella catarrhalis strain O35E employed in these studies does not bind to any CEACAM [Several cellular receptors have already been proposed to act as co-receptors for CEA or to co-operate with epithelial CEACAMs -81. For y CEACAM , it is uIn several studies, it has been observed that CEACAM stimulation has a positive effect on cell-matrix adhesion of epithelial cells as well as on integrin-mediated cell-cell adhesion in leukocytes ,22,82. INeisseria gonorrhoeae, which infects the urogenital tract, it has been observed that the enhanced matrix binding of the infected epithelial cells strongly reduces the exfoliation of the superficial mucosal cell layer [Neisseria meningitidis or Moraxella catarrhalis profits from the presence of epithelial CEACAMs [N. meningitidis, bacteria are not detected in wildtype mice three days after inoculation, whereas the same bacterial strain is present for up to a week in the nasopharynx of CEACAM1-transgenic mice [N. gonorrhoeae, as selective and potent stimuli.Nevertheless, already the indirect connection between CEACAMs and integrins must be advantageous for bacteria trying to get a foothold on the mucosal surface, given the fact that so many unrelated microbes target CEACAMs Table . Indeed,ll layer . Suppresll layer . Further CEACAMs ,92. In tnic mice . It is cOver the last decade, CEACAMs have emerged as important modulators of signaling events in leukocytes, endothelial, and epithelial cells. The simultaneous expression of multiple CEACAM family members by most human epithelial tissues, including GPI-anchored and transmembrane forms in different splice variants, has hampered progress in deciphering the molecular signaling connections initiated by CEACAM-mediated cell-cell interactions. To understand the contribution of CEACAMs in these processes, well characterized antibodies have been employed to interfere with CEACAM-CEACAM interactions, but due to sterical hindrance such approaches might also block a number of other cell-cell interactions. The use of CEACAM-binding bacteria as multivalent, high affinity ligands for a number of epithelial CEACAMs has provided an additional opportunity to selectively trigger CEACAM signaling in vitro and in vivo. These natural probes allow the visualization of local CEACAM-initiated signaling complexes as well as signaling intermediates and, therefore, have provided novel insight. Combining these different approaches will further help to refine our understanding of epithelial CEACAM physiology and of the involved molecular and cellular processes.CEA: Carcinoembryonic antigen; CEACAM: CEA-related cell adhesion molecule; Ig: Immunoglobulin; ITIM: Immunoreceptor tyrosine-based inhibitory motif; PI3K: Phosphatidylinositol-3’ kinase; PTK: Protein tyrosine kinase; TLR2: Toll-like receptor 2.The authors declare that they have no competing interests.AKT and CRH wrote the manuscript. AKT, TS, and CRH revised the manuscript. All authors read and approved the final manuscript.


To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.Elaiophylin is an unusual C Modular type I polyketide synthases (PKSs) are giant multifunctional enzymes, principally from actinomycete bacteria, that use a remarkable assembly-line logic for the biosynthesis of a diverse array of bioactive natural products, includinThe directness of the link between the PKS gene sequence and the chemical structure of the end product has revolutionized our view of the evolution of antibiotic biosynthesis, and has C2-symmetric macrocyclic dilactones, or diolides.[C2-symmetric diolides of diverse ring size have now been characterized from numerous sources including bacteria,[1 b; Scheme An intriguing and relatively rare variation in the mode of polyketide release from modular PKS assembly lines leads to diolides. C2-symmebacteria, fungi,[1bacteria, and maribacteria, A better1 b on the polyketide synthase, as illustrated in Scheme N-acetylcysteaminyl thioesters (SNAC thioesters) of tetraketide and pentaketide analogues of the natural octaketide monomers, which in vivo are acted upon by the TE while tethered to an adjacent ACP domain in the multienzyme assembly line, we show here that the TE can catalyze homodimerization of the synthetic pentaketide 3 b , while pentaketide SNAC thioester 3 b was obtained in 13 steps . In contrast to previously studied TE domains from macrocyclic PKS multienzymes, which retain a dimeric structure,[Candidate substrates for the diolide cyclase were obtained through stereoselective synthesis of analogues of the elaiophylin monomeric seco acid Scheme . Tetrake3 b (3 mm) with Ela-TE (40 μm) in 0.1 m potassium phosphate buffer (pH 8.2) containing 10 % DMSO produced, in a time- and enzyme-dependent manner, the symmetric decaketide diolide 5, the structure of which was confirmed by HRMS and 1D- and 2D-NMR techniques ; as well as the hydrolysis product 3 a. These were accompanied by a further species eluted after 20.5 min . Notably, dimerization of 3 b gave only the symmetrical 16-membered macrodiolide, as found in natural 1 b, with no evidence of regioisomers with a different ring size being formed. We also tested the chain-terminating cyclase/thioesterase from the erythromycin pathway (DEBS-TE)[3 b, but this enzyme exclusively catalyzed hydrolysis to 3 a . These observations show that the TE is competent to catalyze both the ligation of two monomeric polyketide chains and subsequent diolide formation and they support the mechanism of route 2) . Interestingly, when an equimolar mixture of 2 b and 3 b was incubated with Ela-TE under the same conditions, the asymmetric 16-membered macrodiolide nonaketide 6, the structure of which was confirmed by HRMS and 1D- and 2D-NMR , was produced in addition to 5 and in comparable amounts . However insufficient material was available to allow NMR analysis. Further work will thus be required to determine the exact course of the reaction.To the best of our knowledge, this is the first example of a “hybrid” macrodiolide polyketide produced enzymatically in vitro. In this experiment, LC–MS revealed the presence of a species with the mass predicted for a linear nonaketide thioester with the same retention time as Previous structural studies on PKS TE domains catalyzing either hydrolysis or macroThe iterative Ela-TE must catalyze a total of two acylation and two deacylation reactions to form the diolide, and further structural and functional studies will be needed to understand the respective selectivity of these steps. Nevertheless, our present results open the perspective of using such diolide TE domains preparatively to synthesize not only novel macrodiolides but also linear dimeric or even trimeric polyketide esters. Such studies with the purified TE domain of the oxazole diolide conglobatin are unde


Activated Toll-like receptors (TLRs) cluster in lipid rafts and induce pro- and anti-tumor responses. The organization of the assembly is critical to the understanding of how these key receptors control major signaling pathways in the cell. Although several models for individual interactions were proposed, the entire TIR-domain signalosome architecture has not been worked out, possibly due to its complexity. We employ a powerful algorithm, crystal structures and experimental data to model the TLR4 and its cluster. The architecture that we obtain with 8 MyD88 molecules provides the structural basis for the MyD88-templated myddosome helical assembly and receptor clustering; it also provides clues to pro- and anti-inflammatory signaling pathways branching at the signalosome level to Mal/MyD88 and TRAM/TRIF pro- and anti-inflammatory pathways. The assembly of MyD88 death domain (DD) with TRAF3  and TRAF6 (pro-inflammatory) suggest that TRAF3/TRAF6 binding sites on MyD88 DD partially overlap, as do IRAK4 and FADD. Significantly, the organization illuminates mechanisms of oncogenic mutations, demonstrates that almost all TLR4 parallel pathways are competitive and clarifies decisions at pathway branching points. The architectures are compatible with the currently-available experimental data and provide compelling insights into signaling in cancer and inflammation pathways. The signalosome architecture provides the structural basis for TIR-domain signalingThe TIR domain signalosome illuminates receptor clustering upon stimulationAlmost all parallel pathways of TLR4 signaling are competitiveStructural details of interactions reveal the mechanisms of oncogenic mutationsToll-like receptors (TLRs) orchestrate the innate and adaptive immune systems12151821712At almost all levels of TLR signaling, proteins oligomerize to form large multimolecular assemblies23TLR1, TLR2, TLR10, Mal, MyD88, TRAM, TRIF, and IL-1RAPL have resolved structures of their TIR-domains. Among these, TLR10 (PDB_ID: 2j67), C713S mutant TLR2 (PDB_ID: 1o77), and IL-1RAPL (PDB_ID: 1t3g) are in homodimer form. Each TIR domain is composed of five central β-strands (A-through-E) and six surrounding α-helices (A-through-E)6821272930Mutagenesis studies identified interface residues and led to structural models of some of the binary interactions of TIR-domain signalosome682127293032TLRs’ clustering is crucial for efficient signaling, but they cannot form clusters through tetramerization or higher order oligomerization due to steric hindrance of their ectodomains26In our MyD88-dependent signalosome, all TIR domains are in dimer form: a TLR4-dimer recruits two Mal-dimers, which in turn recruit four MyD88-dimers. Different signalosome schemes, with varying stoichiometries of Mal and MyD88 were proposed before: some of them show 2 Mal and 2 MyD88 molecules per TLR4 dimer in the signalosome3639Upon stimulation, TLRs form homo- or hetero-dimers with their Leucine Rich Repeats (LRRs) and TIR domains212221In the first potential TLR4-homodimer model , BB-loopin vivo66Mal has a TIR-domain and a small amino terminal localization domain, through which it can interact with phospholipids, particularly phosphatidylinositol-4,5 bisphosphate P2) that is enriched in lipid raftsLike the TLR4-homodimer, we predicted several architectures for the TLR4-Mal interaction. The two TLR4-dimers (BB and FF) use different interfaces to interact with Mal TIR-domain, suggesting that distinct TLR4-Mal architectures are possible . HoweverMal serves as a bridge between TLR4 and MyD88. Mutational analysis indicated that MyD88 R196 and R288 are at the Mal-MyD88 interface6In order to form the myddosome, comprising MyD88, IRAK4 and IRAK2/IRAK1, MyD88 molecules should dimerize and oligomerize. Although variable stoichiometries have been observed 13Brucella (4lqc.pdb) is also BBAn earlier study suggested more than one binding mode for TIR domain MyD88-dimerization and formation of multivalent aggregatesSimilar to Mal, which is a bridging adaptor and associates with phospholipids in the membrane, TRAM is also a bridging adaptor and is attached to the membrane via its myristoyl groupTRAM links TLRs to TRIF, just like Mal connecting TLRs to MyD88. The C747 residue of TLR4 was shown to be involved in the TLR4-TRAM interface6TRAM homodimer formation is required for TRIF recruitment. TRAM acts as a scaffold bringing TLRs and TRIF together. This assembly is a key upstream branching step in the interferon and anti-inflammatory pathway . Monomer1821Clustering of MyD88 DDs initiates the oligomerization of the myddosome complex. Besides IRAK4 and IRAK2, MyD88 DDs also associate with TRAF64715Endosomal TLRs can signal through both TRAF3 and TRAF6. TRAF6 signaling activates the classical NF-κB pathway, leading to expression of pro-inflammatory cytokines20FADD is another protein interacting with MyD88 DD47Remarkably, the C27* nonsense mutation on FADD protein, which is clinically observed in lung squamous carcinoma with 0.21 frequency (according to the TCGA data)Taken together, the TLR4 architectures indicate that all TLR’s parallel downstream pathways are competitive . MyD88- To conclude, considerable effort has been invested in the quest for the entire TIR signalosome assembly, including its clustered architectures. This problem is significant since TLR activation involves clustering and signalosome formation. As we show here, the architectures may clarify TLR4 physiological signaling control and how it can go wrong in disease. Here, we present the two, MyD88-dependent and TRIF-dependent TIR-domain signalosome assemblies. We exploit experimental mutational data in every step in the construction. TLR activation through its ectodomain dimerization can elicit the proinflammatory, anti-viral and anti-inflammatory, and apoptosis pathways. Binding to Mal/MyD88 or TRAM/TRIF is the step making the first cellular decision. Our results suggest that steric hindrance of Mal and TRAM in a TLR/TRAM/TRIF assembly and Mal/MyD88 and TRIF leads to competitive binding to TLR’s TIR domain. Our results further reveal that parallel downstream pathways with opposing consequences are competitive at almost each branching point of the TLR pathway, beyond Mal and TRAM recruitment. TRAF6, TRAF3, and FADD, whose recruitment results in pro-inflammatory, anti-inflammatory, and death pathways respectively, present similar scenarios. Our signalosome architectures with 8 MyD88 molecules are important, since they provide the basis for obtaining an insight into how TLR4 clusters. The 8 MyD88 molecules connect into multiple 6 MyD88 molecules myddosome helical signaling units, and unveil downstream oligomerization clusters formed by stimulated TLRs. We speculate that the long MyD88 linker is critical for TLR clustering, and could be its raison d'être. The clusters, and their multivalent network, particularly through TRAFs58We obtained the upstream TIR-domain interactions and downstream DD interactions based on binary interactions of proteins predicted by PRISM32in silico mutagenesis by using the FoldX plugin for the YASARA molecular viewerMutations of the proteins in the TLR network are obtained from cBioPortal for Cancer Genomics How to cite this article: Guven-Maiorov, E. et al. The Architecture of the TIR Domain Signalosome in the Toll-like Receptor-4 Signaling Pathway. Sci. Rep.5, 13128; doi: 10.1038/srep13128 (2015).