Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila  and its multidomain immunity protein  have been done. xcinA-ximB , translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10 kDa) protein complex had toxic activity. Atypical cognate immunity protein (42 kDa) of xenocin was fusion of immunity domain (10 kDa) and hemolysin domain (32 kDa). In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes. Bacteriocins are toxins produced by the bacteria to inhibit the growth of similar or closely related bacterial strain(s) during stress conditions . They arXenorhabdus nematophila is a motile gram-negative bacterium belonging to the family Enterobacteriaceae, and forms a symbiotic association in the gut of soil nematode from the family Steinernematidae [X. nematophila can be grown under standard laboratory conditions. Growth in vitro is probably supported by the rich nutrient supply of the laboratory growth media and lack of competition that normally exists in the soil environment. As the bacteria enter stationary phase of growth cycle, they secrete several extracellular products, which include lipase(s), phospholipase(s), and protease(s), and several broad spectrum antibiotics that can be assayed in the culture media [X. nematophila known as xenocin [Bacillus, Enterobacter, Enterococcus, Citrobacter, Serratia, and Stenotrophomonas [X. nematophila is a fusion of two different domains, immunity domain and hemolysin domain. (4) Immunity protein has ATPase activity, although Walker motif is missing in its primary amino acid sequence. (5) Xenocin-immunity protein complex is secretory in nature without any signal sequence.ematidae , 7. Freeematidae . Bacteriematidae , 9. X. nre media , 11. There media . In our  xenocin . Recombiphomonas . Xenocinphomonas . (2) Thephomonas . (3) Sizphomonas . ImmunitxcinA and ximB genes. Neutralization of xcinA toxicity by immunity protein domain is determined by endogenous assay. Exogenous toxicity assays were performed with purified recombinant xenocin-immunity domain protein complex and other domains. In silico study of the immunity protein showed its similarity with hemolysin and purine NTPase like protein; therefore, hemolysis and ATPase assays were performed. Finally, secondary structural analysis of recombinant xenocin-immunity domain protein complex, catalytic-immunity domain protein complex, immunity protein, and its hemolysin domains were performed with circular dichroism.In this study we have cloned, expressed, and purified all the possible domains of E. coli strains DH5α (Bethesda Research Laboratories) were used as the host for cloning. E. coli BL 21(DE3) pLysS strain from Novagen and M 15 strain from Qiagen were used in the expression studies. The plasmid vector pGEM-T Easy from Promega  were used for PCR cloning. LB medium was used for growing bacterial strains. Ampicillin, Kanamycin, and Chloramphenicol were used in the concentration of 100, 35, and 25 μg mL−1, respectively.All the chemicals were purchased from Sigma (Sigma-Aldrich) except where otherwise mentioned. Ligase and endonucleases were purchased from Promega . Vector pGEM-T easy was procured from Promega . Vector pQE30, Ni-NTA agarose resin and QIA quick spin columns were from Qiagen (Germany). Oligonucleotides were custom synthesized by Sigma. Phylogenetic analysis of immunity protein was done by a method as described earlier . BrieflyX. nematophila. The ORF 1 encoding xcinA gene and partial ORF 2 encoding immunity domain of ximB gene were obtained by PCR amplification using primer 1 with a BamHI site at the 5′ end and a reverse primer 6 with HindIII site at the 3′ end. The amplified product (2 kb) was ligated in pGEM-T Easy and pQE30 vector, producing pJC5 and pJC6 plasmids, respectively.Primer pairs used for cloning studies are shown in xcinA gene was cloned with the immunity domain (first 270 bp of ximB gene) of ximB gene using primer pair 2 and 6 as described earlier [The catalytic domain (318 bp) of  earlier .BamHI site at the 5′ end and a reverse primer TransB with NcoI site at the 3′ end were used to amplify the translocation domain of xcinA gene. Amplified product of 1 kb was ligated in pGEM-T Easy vector and pQE30 vector, producing pJC7 and pJC8 plasmids, respectively.Primer 1 with a BamHI site at the 5′ end and a reverse primer RecB with HindIII site at the 3′ end were used to amplify the translocation-receptor domain of xcinA gene. The amplified product (1.5 kb) was ligated in pGEM-T Easy vector and pQE30 vector producing pJC9 and pJC10 plasmids, respectively.Primer 1 with a BamHI site at the 5′ end and a reverse primer 5 with HindIII site at the 3′ end used for ximB cloning and forward primer 4 without any restriction site and backward primer 7 without any restriction site were used for cloning ximB hemolysin domain. The amplified products of 1 kb and 700 bp were ligated in pGEM-T Easy vector producing pJC11 and pJC12 plasmids. The 1 kb amplified product was further ligated in pQE30 and 700 bp amplified product was ligated in pQE31 expression vector producing pJC13 and pJC14 plasmids, respectively.Primer 3 with xcinA and immunity domain of ximB gene with native promoters was amplified using XenocinF1 (300 bp upstream of start codon of xcinA locus) and primer 6 and cloned in pGEM-T Easy vector producing pXIM construct. The empty pGEM-T Easy vector designated as pGEM was used as control. Both plasmids were transformed in DH5α cells. The XIM and GEM strains were used for endogenous assays. All the constructs and strains are listed in A 2.330 kb DNA fragment containing both  E. coli BL 21(DE3) pLysS cells. The resulting strains JC4, JC6, JC8, JC10, JC13, and JC14 were used for expression and purification of recombinant proteins under the control of IPTG inducible T7 promoter as per the protocol described earlier [600 reached 0.5. Culture was induced by adding 1 mM final concentration of IPTG and incubated at 30°C for 6 hours. Cells were harvested and washed with 40 mL of cold and 50 mM sodium phosphate buffer, pH 8, containing 300 mM NaCl and 50 mM benzamidine (buffer A). The cell pellet was suspended in 25 mL of buffer A and cells were disrupted by sonication at 4°C. The cell lysate was centrifuged at 12000 ×g for 30 min at 4°C in a RC5 plus centrifuge, and the 6XHis-tagged recombinant proteins or protein complexes in the soluble fractions were purified as follows. The supernatant from the previous step was loaded on Ni-NTA agarose column preequilibrated with buffer A at 4°C. The column was washed extensively with buffer A, containing 25–50 mM imidazole, and the protein/protein complex was eluted with buffer A containing 300 mM imidazole. Fractions containing pure protein or protein complex were concentrated using centricon (Millipore PM10). Recombinant protein or protein complexes were dialyzed overnight against 100 volumes of 50 mM sodium phosphate buffer, pH 8, and the final preparations were stored at −20°C in the presence of 15% glycerol.The plasmids pJC6, pJC8, pJC10, pJC13, and pJC14 were transformed in M15 cells where as pJC4 was transformed inxcinA gene was cloned with its native promoter along with immunity domain of ximB gene which gave rise to pXIM. This construct was transformed in E. coli DH5α to give rise to XIM strain. Empty pGEM-T Easy vector was considered as control and transformed in E. coli DH5α to give rise to GEM strain. Overnight grown strains GEM and XIM were subcultured in fresh medium and incubated till the OD600 reached 0.5. The cultures were diluted in fresh medium 1 : 100 and induced with 0.3 μg mL−1 of mitomycin C (an inducer of xenocin native promoter). The optical densities of the cultures were monitored at 600 nm during different intervals.To study the neutralizing effect of the immunity domain protein, E. coli DH5α strain grown in M9 medium, and the protein complex was applied to sterile disks. The plates were incubated overnight at 37°C, and the sizes of clearance zones were recorded.The bacteriostatic activity of purified recombinant proteins/complexes were determined by the protocol as described earlier . BrieflyμL) of protein (5 μM) sample dissolved in PBS and erythrocytes suspension were added into wells of 96-well plate. PBS and water was used to establish 0 and 100% hemolysis, respectively. The plate was then incubated at 37°C for 1 hr and centrifuged at 1000 ×g for 5 minutes. The resulting supernatant was transferred to new wells, and the absorbance was determined at 540 nm on a continuous spectrum microtitre plate reader.Freshly isolated rabbit blood cells were washed thrice with phosphate buffer saline (PBS) by centrifuging at 1000 ×g, 4°C for 10 minutes. Washed erythrocytes were resuspended in PBS to make a final concentration of 4%. The same volume  in a buffer containing 20 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 100 mM KCl, 8 mM DTT, and 80 μg/mL of BSA in a total reaction volume of 10 μL. Samples were incubated at 37°C for 30 minutes. At the end of the reaction, 1 μL of the reaction mixture was spotted on a polyethyleneimine thin-layer chromatography (TLC) plate  and air-dried. Chromatography was performed using 0.5 M LiCl and 1 M HCOOH as the running solvent. The TLC paper was air-dried and autoradiographed.ATPase assay was performed with recombinant immunity protein and its domains. Protein samples of different concentrations were incubated with 0.2 μL sample volume) on a Jasco J-810 spectropolarimeter equipped with a Jasco Peltier temperature controller at 25°C using 1 mm optical path length quartz cells and the step size was 0.5 nm with 1 nm bandwidth at a scan speed of 20 nm minute−1. Averages of 5 scans were obtained for blank and protein spectra, and the data was corrected for buffer contribution. Measurement was performed at protein concentration of 1 μM under nitrogen flow. The secondary structure percentages were calculated using K2d computer modelling program. The results were expressed as mean residue ellipticity in units of degree/cm2/dmol1.The far-UV CD spectrum was recorded between 190 and 260 nm  of ximB gene. Purification of recombinant xenocin-immunity domain (10 kDa) protein complex from JC6 strain was done by Ni-NTA chromatography under native conditions. Two bands were visible in SDS-PAGE. One was at the position corresponding to 66 kDa and another below 14 kDa protein marker as shown in xcinA and immunity domain of ximB as shown in xcinA gene from JC8 strain with Ni-NTA chromatography showed multiple bands in SDS-PAGE as shown in Phylogenetic analysis of the xenocin  and its xcinA by immunity domain of ximB gene was determined by endogenous assay. Endogenous assay with XIM strain (harboring pXIM containing xcinA with its native promoter and first 85 amino acid residues of ximB gene) and GEM strain (harboring empty pGEM T-Easy vector) in the presence of mitomycin C showed the same growth profile as shown in Neutralization of endogenous toxicity of E. coli DH5α as target cells. The zone of inhibition was observed as shown in ximB gene or its hemolysin domain (32 kDa) was used for exogenous assay as shown in Exogenous toxicity assay was performed with purified recombinant xenocin-immunity domain (10 kDa) complex using http://www.ncbi.nlm.nih.gov/BLAST/) of the immunity protein showed its similarity with hemolysin (AAF42109) and purine NTPase like protein (data not shown). Hemolytic assay with fresh rabbit red blood cells was performed with purified full length immunity protein as well as its immunity and hemolysin domain. Results showed that none of the protein had hemolytic activity (data not shown).Protein-protein blast results  protein complex, catalytic-immunity domain protein complex, immunity, and its hemolysin domain were recorded at 25°C as shown in X. nematophila forms a distinct cluster in phylogenetic tree [X. nematophila forms a separate cluster in the very beginning. This could be due to variable length of immunity protein from X. nematophila. Cognate immunity protein of xenocin consists of 368 amino acid residues and is a unique fusion of two different domains. Its N′ terminal (first 85 amino acid residues) showed similarity with immunity protein from other prokaryotic systems, whereas the C′ terminal showed similarity with hemolysin (N. meningitidis accession no. AAF42109) and purine NTPase like proteins.Recombinant xenocin-(66 kDa-) immunity (42 kDa) protein complex has a broad range bacteriostatic property, inhibiting the growth of six insect gut resident bacterial species . Due to tic tree . PhylogeThree-dimensional structure of xenocin has been recently deciphered by homology modelling in my lab . It is axcinA gene alone in expression vector, not a single transformant was observed. One reason for this result could be the leaky expression of toxic xcinA gene. To address this question, xcinA gene was cloned along with N′ terminal immunity domain (10 kDa) of ximB gene. When the recombinant protein from JC6 strain was purified by Ni-NTA chromatography, xenocin was visible in SDS-PAGE at position corresponding to 66 kDa whereas immunity domain of immunity protein was observed below 14 kDa protein marker. This result showed that N′ terminal immunity domain (10 kDa) of immunity protein (42 kDa) encoded by ximB gene is enough to bind with and neutralize the in vivo toxic effect of xcinA gene. To confirm this result and to map the minimum functional domain of immunity protein required to abolish the xcinA gene toxicity in vivo, first 85 amino acids of immunity protein were cloned along with xcinA gene under its native promoter. Same growth profile of XIM strain (harboring pXIM) and GEM strain (harboring empty pGEM T-Easy vector) in the presence of mitomycin C confirmed that first 85 amino acid residues of ximB gene were able to neutralize the toxic activity of xenocin in vivo. Further, purification of recombinant catalytic-immunity domain protein complex with Ni-NTA chromatography under native conditions from JC4 strain [xcinA and ximB genes that could be expressed and purified.While cloning 4 strain  confirmexcinA gene alone which is composed of translocation, receptor, and catalytic domain an attempt had been made to clone, express, and purify the translocation domain alone or along with receptor domain of xcinA gene. In native conformation, translocation domain of bacteriocin like colicin E3 interacts with catalytic domain of E3 and immunity protein via receptor domain, and this interaction further provides stability to the translocation domain [xcinA, receptor and catalytic domains as well as immunity protein were missing which may be probably made it to attain an open conformation and be susceptible to proteases from the host cells. Hence, during the purification of translocation domain from pJC8, multiple bands were observed in SDS-PAGE. However, western blot with purified fraction when probed with anti-His antibody showed a single band corresponding to the size of translocation domain which confirmed the expression of translocation domain alone. Further, purification of recombinant translocation-receptor domain protein with Ni-NTA chromatography from JC10 strain, showed two prominent bands in SDS-PAGE rather than multiple bands. Upper band corresponded to ~52 kDa whereas lower band corresponded to ~28 kDa. western blot using whole cells and purified fraction when probed with anti-His antibody showed a single band at 52 kDa which is corresponding to a size of recombinant translocation-receptor domain protein. Therefore, we inferred that recombinant translocation domain along with receptor domain of the xcinA gene was more stable as compared to the recombinant translocation domain alone, but due to the absence of catalytic domain and immunity protein it was still prone to proteases of the host cell.As we were unable to express n domain . HoweverxcinA toxicity. Results showed that first 85 amino acids of the immunity protein were able to neutralize xenocin endogenous toxicity as both strains had the same growth profile. Moreover, from this experiment we inferred that expression of cognate immunity protein domain and its binding to the catalytic domain of xenocin occurs in the host cell simultaneously.Endogenous toxicity assays with XIM and GEM strains were performed to identify the minimum domain required to neutralize the X. nematophila is a unique fusion of immunity domain (10 kDa) and hemolysin domain (32 kDa) protein, and endogenous assay confirmed that immunity domain of ximB gene is enough to neutralize the detrimental effect of xcinA gene. Therefore, exogenous assay was performed with purified recombinant xenocin-immunity domain (10 kDa) complex using E. coli DH5α as target cells to determine translocational ability of xenocin-immunity domain (10 kDa) complex into the cytoplasm of target cells. Clear zone of inhibition was observed in the exogenous toxicity assay due to the detrimental effect of xenocin-immunity domain (10 kDa) protein complex. This result confirmed the functionality of translocation as well as receptor domain of xenocin and their individual roles in internalization of xenocin into the cytoplasm of target cells. Moreover, it also confirmed that hemolysin domain (32 kDa) of ximB gene has no role in the internalization of xenocin as well as in exogenous toxicity.Receptor domain in bacteriocin plays a major role in the exogenous toxic effect, as it is the first domain which binds to ligand present on the surface of its target cells. Binding is followed by import of bacteriocin into the cells with the assistance of either Ton proteins  or Tol proteins  of periplasm and facilitate the translocation of the catalytic domain of bacteriocins into the periplasmic space of the target cell for further processing , 16–18. xcinA gene along with immunity domain of ximB gene was the minimum domain which could be expressed and purified. Therefore, purified catalytic-immunity domain protein complex was studied for the bacteriostatic effect in the exogenous assays. Results showed that zone of inhibition was not observed, which again confirmed the role of translocation as well as receptor domain of xcinA gene for its internalization into the cytoplasm on the target cells. Similar results were observed when full length immunity protein (42 kDa) encoded by ximB gene or its hemolysin domain (32 kDa) was used for exogenous assay. Moreover, as expected zone of inhibition was not observed in the experiment in which buffer was used as a negative control.Catalytic domain of ximB gene corresponds to 42 kDa. Protein-protein blast using BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/) of the immunity protein showed similarity with hemolysin (AAF42109) and purine NTPase like protein. Hemolytic assay with fresh rabbit red blood cells was performed with purified full length immunity protein as well as its immunity and hemolysin domains. Due to the partial primary amino acid sequence similarity with hemolysin protein, none of the protein/protein domains showed hemolytic activity.To neutralize the detrimental effect of bacteriocins, they are always expressed along with cognate immunity protein . The molin silico analysis did not show Walker motif in its primary amino acid sequence, which is generally present in the proteins having ATPase activity [As immunity protein also showed similarity with protein like purine NTPase's ATPase assay was performed with purified recombinant full length immunity protein, its immunity domain, and hemolysin domain. Although full length immunity protein showed ATPase activity, activity . The rolα-helical structure of xenocin-immunity complex is attributed due to the helix turn helix structure of receptor domain of xenocin which corroborates with its recently deciphered three-dimensional structure [β-sheet and only 7% α-helical structure. This open conformation with high β-sheet might be beneficial to bind with its substrate (RNA) and act upon it [α-helical where as 13% was β-sheet. Hemolysin domain of the immunity protein also had the same secondary conformation with 30% α-helical and 11% β-sheet content. Since immunity protein is a novel fusion of two different domains the role of its secondary is not deciphered yet.Secondary structure analysis of purified recombinant xenocin-immunity domain (10 kDa) protein complex, catalytic-immunity domain protein complex, immunity, and its hemolysin domain was done with far UV spectra. High percentage of tructure . Catalyt upon it , 21. In


Lymphatic vessel dysfunction is associated with various pathologic conditions, including immunologic disorders, lymphedema, as well as tumor dissemination. Yet, the knowledge about the regulation of lymphatic vessel development is still limited. Our study elucidates cyclin dependent kinase 5 (Cdk5) as an essential player in the development of lymphatic vessels. Deletion of Cdk5 in the mouse endothelium results in severe lymphedema formation and embryonic lethality. On the mechanistic level, we show that Cdk5 phosphorylates the forkhead transcription factor Foxc2 which regulates Foxc2-dependent transcription. In summary, our study elucidates the Cdk5-Foxc2 interaction as a critical regulator of lymphatic vessel development. The lymphatic system is essential for the maintenance of tissue fluid homeostasis, enables the uptake of dietary lipids and regulates the immune response by serving as a trafficking route for immune cells . LymphatMalfunctioning of the lymphatic system is associated with various pathologic conditions. Dysfunction of the lymphatic vasculature results in lymphedema formation and compromises immune function. Moreover, the lymphatic system contributes to tumor cell metastasis – tumors induce lymphangiogenesis to allow the dissemination of tumor cells .The forkhead transcription factor Foxc2 plays a central role in lymphatic vessel development and lymphatic valve formation -6. Mutatin vivo function of Cdk5 in the endothelium was still missing.Our recent study elucidates the serine-threonine kinase cyclin dependent kinase 5 (Cdk5) as the missing link in the regulation of Foxc2 in the lymphatic endothelium . The “neBy using endothelial-specific Cdk5 knockout mouse models, our recent work demonstrated that Cdk5 is essential for lymphatic vessel development. Endothelial-specific deletion of Cdk5 causes congenital lymphatic dysfunction with lymphedema and finally results in embryonic lethality. Cdk5 knockdown embryos develop non-separation of blood and lymphatic vessels and show severe defects in lymphatic valve formation and lymphatic vessel patterning with ectopic coverage of lymphatic vessels by smooth muscle cells. As the underlying mechanism, we identified Cdk5 as the upstream kinase of Foxc2. Cdk5 phosphorylates Foxc2 and activates Foxc2-dependent transcription. As a consequence, Cdk5-mediated phosphorylation of Foxc2 regulates the expression of Foxc2-dependent target genes. Among others, connexin 37, a downstream target gene of Foxc2 that is crucial for lymphatic valve formation , 18, wasCollectively, our findings elucidate the Cdk5-Foxc2 interaction as a critical regulator of the transcriptional network underlying lymphatic vascular remodeling and thereby provide novel insight into the regulation of lymphatic vessel development. Moreover, because Cdk5 represents a drugable kinase , 17, our


This first structure of the enzyme converting dopamine to norepinephrine provides new perspectives on numerous disorders. The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson’s disease, schizophrenia, Alzheimer’s disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON  domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system. Dopamine β-hydroxylase  (DBH) catalyzes the hydroxylation of dopamine to norepinephrine  monooxygenase (PHM)  domain, which belongs to the class of DOMON-like domains  293S cells is present both as a dimer and a tetramer, which can be separated by size exclusion chromatography. The dimer and tetramer do not interconvert in the pH interval 4 to 9 (see figs. S4 to S7). However, under denaturing conditions, the tetramer converts to a dimer, and upon addition of a reducing agent, the dimer converts to a monomer (see fig. S8). Crystallization experiments gave diffraction quality crystals of the dimeric form.H and CuM domains, and the C-terminal dimerization domain.The overall three-dimensional structure of dimeric human DBH is shown in H and CuM domains) has the same topology as the structure of PHM (M domain and a β-strand (residues 608 to 614) that is part of the DOMON domain, creating a very integrated structure. This is illustrated in the secondary structure organization of DBH shown in The DOMON domain has an immunoglobulin (Ig)–like β-sandwich structure, the catalytic core  of 0.71 Å for 149 atoms, alignment of the catalytic CuH domain A on CuH B gives an RMSD of 1.15 Å for 137 atoms, and alignment of the catalytic CuM domain A on CuM B gives an RMSD of 0.69 Å for 169 atoms. The dimerization domain does, however, not overlay very well with itself (RMSD of 3.83 Å for 53 atoms). Omitting the loop/helix from residues 525 to 538 improves the alignment, and the RMSD becomes 0.87 Å.The dimeric structure is asymmetric. In the A chain, the two catalytic Cu-sandwich, containing five and six antiparallel β-strands, respectively, as shown in Phanerochaetechrysosporium cellobiose dehydrogenase (CDH) [Protein Data Bank (PDB) ID 1D7B] and the Aromatoleum aromaticum ethylbenzene dehydrogenase α subunit (PDB ID 2IVF) and, to a lesser extent, the carbohydrate-binding module from Thermotoga maritima xylanase (10ACBM9-2) (PDB ID 1I8A). Structural alignment of the DOMON domain in DBH with the cytochrome domain of CDH shows an identical fold of the two domains  in the vicinity, are conserved among DBH DOMON domains from different organisms (see fig. S13). In the structure, we have placed water molecule 5 in chain A; however, in chain B, the electron density did not support modeling of an extra water molecule. On the basis of the very oxygen-rich ligand environment, it is likely to be either an alkali metal ion or an alkaline earth metal ion . However, a search using the CheckMyMetal server did not reveal the possible identity of the metal, most likely because not all the ligands are prealigned for metal binding.Behind the possible ligand-binding pocket appears to be a metal ion–binding site coordinated by Asplose see . These f154 and C596. It also contains two glycan sites at Asn64 and Asn184. Both of them could be built in chain A, whereas in chain B, the electron density only allowed for building of the glycan at Asn64.The DBH DOMON domain is linked to the C-terminal part of the protein via a disulfide bridge between CH binds and a C-terminal domain where CuM binds. Both domains consist primarily of β sheets and have the approximate dimensions of 37 × 45 × 33 Å and 44 × 45 × 33 Å for chains A and B, respectively. Both domains have the same topology as described for PHM  and strand β2 with β10 (C390-C503), respectively. The C-terminal sheet is actually a six-stranded sheet by the addition of a β-strand made of residues from the C terminus of the protein (residues 561 to 566) following the dimerization domain, as described above. The domain is further stabilized by a disulfide bridge to the additional strand C394-C565  in the dimerization domain of molecule A makes hydrophobic interactions with residues 376 to 379 and 478 in the CuM domain, and the second β-strand is part of the DOMON domain  Build 142], and they are distributed over the entire sequence; however, none of the copper ligands or the glycosylation sites are affected.DBH is found in vesicles of central adrenergic and noradrenergic neurons, as well as peripheral noradrenergic neurons, and is released to the blood in response to stimulation [(Inhibitors of DBH (nepicastat and etamicastat) are currently in clinical development for treatment of cocaine dependence  of the monomer is different from our findings. The crystal structure also shows a much more integrated structure.A comparison of the overall structure of the DBH monomers with the model structure published by Kapoor et al. , residues 40 to 617 , was optimized for expression in HEK293 cells and synthesized by GenScript Corporation. For cloning into the pHLsec expression vector . DBH was eluted with 5 mg of FLAG tag peptide per milliter in 10 mM Hepes and 150 mM NaCl (pH 7.5) and concentrated to 2 ml by ultrafiltration. DBH was present both as a tetramer and a dimer, which were separated on a Superdex 200 HR 16/60 in 10 mM Hepes and 150 mM NaCl (pH 7.5) at 1 ml/min. Fractions containing the dimeric DBH were collected and pooled; the final concentration that was also used for crystallization experiments was 6 mg/ml.Cells were removed by centrifugation at 5000SeMet labeled protein was expressed and purified as described above with the exception that the medium was exchanged every day and that the cells were harvested after 3 days. The yield was about 10% of that in normal medium.− cells was crystallized using the high-throughput facilities at Oxford Protein Production Facility  ethylene glycol/reservoir solution before flash-freezing them in liquid nitrogen.Glycosylated human DBH dimer FLAG-tagged and expressed in HEK293S GnTi2PtCl4 (λ = 1.0714 Å), also collected at I24. All data were processed with xia2 ; native 2, collected at I24 (λ = 1.0071 Å); SeMet, collected at I24 (λ = 0.9789 Å); and K89 was used as a marker for the sequence together with the glycosylated Asn64 with the neighboring Trp63. In the catalytic domains, the amino acid sequence was changed according to a structural alignment. The initial refinement was performed in refmac5 (M atom was inserted—it is weakly occupied, though, and could just as well be modeled with a water molecule. All together, only 13 water molecules were inserted on the basis of the difference density. Some parts of the structure could not be modeled because of missing or inadequate electron density. The following residues were missing: A40 to A45, A109, A593 to A607, and A612 to A617; and B40 to B46, B104 to B109, B273 to B275, B288 to B291, B597 to B608, and B615 to B617. The quality of the final structure was evaluated with MolProbity (The DOMON and the C-terminal domains were built manually in Coot because all attempts to perform automatic building failed. MetAll molecular graphics were prepared using PyMOL (


Summary: PPDMs is a resource that maps small molecule bioactivities to protein domains from the Pfam-A collection of protein families. Small molecule bioactivities mapped to protein domains add important precision to approaches that use protein sequence searches alignments to assist applications in computational drug discovery and systems and chemical biology. We have previously proposed a mapping heuristic for a subset of bioactivities stored in ChEMBL with the Pfam-A domain most likely to mediate small molecule binding. We have since refined this mapping using a manual procedure. Here, we present a resource that provides up-to-date mappings and the possibility to review assigned mappings as well as to participate in their assignment and curation. We also describe how mappings provided through the PPDMs resource are made accessible through the main schema of the ChEMBL database.Availability and implementation: The PPDMs resource and curation interface is available at https://www.ebi.ac.uk/chembl/research/ppdms/pfam_maps. The source-code for PPDMs is available under the Apache license at https://github.com/chembl/pfam_maps. Source code is available at https://github.com/chembl/pfam_map_loader to demonstrate the integration process with the main schema of ChEMBL.Contact:jpo@ebi.ac.uk Systematic analyses of bioactive small molecules and their molecular targets and homologues form the basis of a number of novel applications in computational drug discovery and systems and chemical biology, including methods of target prediction , see A successful mapping if exactly one of the Pfam-A domain models from the catalogue matches the sequence.No mapping if none of the Pfam-A domain models from the catalogue match the sequence;A conflicting mapping if multiple domain models from the catalogue match the sequence.The objective of the mapping heuristic is to annotate biological assays reported in ChEMBL with the protein domain that mediates small molecule binding. The heuristic is based on protein domain annotations provided through the Pfam-A collection of sequence-based protein domains  can be downloaded from the evidence section. For integration of mappings assigned using PPDMs into the main schema of the ChEMBL database, a standardized procedure exists. Prior to each ChEMBL release, the most recent version of the catalogue is obtained from PPDMs. In a second step, it is applied to proteins that are defined as targets in assays meeting the required criteria. Finally, the set of manually assigned mappings is obtained from the PPDMs resource and used to override mappings that have been assigned by the default procedure.PPDMs provides a richer, domain-level perspective of small molecule binding and enriches annotation of small molecule bioactivities stored in the ChEMBL database. We anticipate that this type of annotation will improve the precision of target prediction and efficacy modelling approaches, and interpretation of the effects of natural genetic variation. PPDMs enables the refinement of domain-level annotation of small molecule bioactivities in a facile and transparent manner. The curation of conflicting mappings in PPDMs is ongoing and we are hopeful that PPDMs can engage the community in reviewing and improving domain-level annotations of small molecule bioactivities.


Bacteroides thetaiotaomicron, suggesting their involvement in polysaccharide metabolism, but little else is known of the function of this domain.Gut microbiome metagenomics has revealed many protein families and domains found largely or exclusively in that environment. Proteins containing the GxGYxYP domain are over-represented in the gut microbiota, and are found in Polysaccharide Utilization Loci in the gut symbiont Genomic context and domain architecture analyses support a role for the GxGYxYP domain in carbohydrate metabolism. Sparse occurrences in eukaryotes are the result of lateral gene transfer. The structure of the GxGYxYP domain-containing protein encoded by the BT2193 locus reveals two structural domains, the first composed of three divergent repeats with no recognisable homology to previously solved structures, the second a more familiar seven-stranded β/α barrel. Structure-based analyses including conservation mapping localise a presumed functional site to a cleft between the two domains of BT2193. Matching to a catalytic site template from a GH9 cellulase and other analyses point to a putative catalytic triad composed of Glu272, Asp331 and Asp333.We suggest that GxGYxYP-containing proteins constitute a novel glycoside hydrolase family of as yet unknown specificity. The era of pyrosequencing has shed light on new areas of the protein sequence universe, revealing new domains and expanding membership of existing domains,2. One pBacteroides is a genus of Gram-negative bacteria, particularly prominent in the distal gut of mammals, including humans, and typically making up to 30% of the microbiota and GxGYxYP_C [Pfam:PF14323]). Three conserved acidic residues are arranged in a similar manner as those comprising the catalytic site of unrelated cellulases and suggest that the GxGYxYP domain defines a new family of glycoside hydrolase (GH).Here we apply wide range of bioinformatics methods, including structure-based analyses of a newly determined crystal structure, to predict a function for the GxGYxYP domain, found in four PULs in Interestingly, proteins bearing the GxGYxYP domain are highly over-represented in human gut metagenomics samples: there are currently around seven times as many such sequences in the MetaHIT database as in UnCapsaspora owczarzaki and the choanoflagellates Monosiga brevicolis and Salpingoeca strain ATCC 50818. There is a single archaeal sequence, from Pyrobaculum arsenaticum, in the present database. The single sequence from moss (Physcomitrella patens subsp. patens) [UniProt:A9U7X7] is annotated as a fragment. The genome of moss is a draft genome and limited extension of the protein sequence is possible at the N-terminus. This extension is most similar to sequences from Paenibacillus so it is possible that the moss sequence is a contaminant. The sporadic eukaryotic distribution does not suggest that this domain was found in the common ancestor of bacteria and eukaryotes. Indeed, phylogenetic analysis provides strong evidence for acquisition of the domain by eukaryotes through multiple instances of lateral gene transfer. As Figure Herpetosiphon aurantiacus sequence. Similarly, the Capsaspora sequence resides in a clade bounded by the bacterial Chitinophaga pinensis sequence.The distribution of a protein family often provides clues as to its function. We therefore analysed the set of species bearing GxGYxYP domains and carried out bootstrapped phylogenetic analysis on the set of full-length GxGYxYP obtained by database searching. The distribution of GxGYxYP-containing species is largely bacterial, with Proteobacteria, Firmicutes and Actinobacteria all represented. Interestingly, however, the domain is seen sporadically in diverse eukaryotes such as B. thetaiotamicron, a prominent commensal gut bacterium. Along with other Bacteroides species it can process a large number of different polysaccharides, both plant compounds eaten by the host and host-derived complex carbohydrates. Upon exposure to carbohydrates, appropriate sets of metabolic enzymes are induced from genes arranged in Polysaccharide Utilization Loci (PULs). Intriguingly, genes encoding GxGYxYP proteins in Bacteroides thetaiotamicron are found in PULs, implicating them broadly in carbohydrate metabolism  resulting in the release of phosphate. This doBacteroides thetaiotaomicron VPI-5482 was determined to 1.25 Å resolution by the MAD method. Data collection, model and refinement statistics are summarized in Additional file3 Da-1 and the estimated solvent content is 43.1%. The Ramachandran plot produced by MolProbity, polysaccharide deacetylases eg [PDB:3rxz] and glucanotransferases eg [PDB:1k1x]. However, the sequence identity shared by matching regions did not exceed 15% and was generally much lower. Furthermore, analysis of the structure alignments revealed that in no case were key catalytic residues found in matching structures present in BT2193. Sequence-based analysis gave similar results. Thus, although families with barrel domains such as Carbohydrate Esterase family 4 were reliably matched by sensitive Hidden Markov Model comparisons using HHpred,37, sequIn broad terms, the 7-stranded β/α barrel is weakly suggestive of a role for BT2193 in carbohydrate metabolism. The SCOP database, for exaSuperficially, these subdomains resemble the Rossmann fold yet that structure was not prominently featured in DALI results for the third subdomain which shares its four β/α construction. The top hit for the third domain (with a Z score of 5.8) was in fact a periplasmic binding protein which contains a central six-stranded sheet. A minimal, artificial Rossmann fold [PDB:2kpo] was the top structural match (Z score 4.2) of the first subdomain yet there was again a fundamental mismatch between the three-stranded central sheet of the BT2193 sub-domain and the four-stranded Rossmann fold. Overall, the results reveal no clear homology between the repeating subdomain of the N-terminal domain and any known structure.3, significantly larger than the next largest cavity with a volume of 1341 Å3. Within the cavity, Profunc also picks out a nest structure[Binding proteins typically interact with their ligands at their largest cavities. Cavity tructure from Asptructure. Also intructure. Figure A further powerful method for prediction of functional sites is picking out examples of local convergent structural evolution. For example, the Ser-His-Asp catalytic triad originally characterised in trypsin-like proteases has since been discovered, along with variant forms such as Ser-His-Glu, in diverse folds with protease, lipase, acyltransferase and general esterase activities eg-47. MoreIf the identified acidic residues in BT2193 were indeed a site for glycoside hydrolase activity then the expectation would be that neighbouring surfaces would bind other monosaccharides in the substrate conferring specificity and/or binding affinity. In glycosidases a minimum site would bind a disaccharide, but endo-acting glycanases, which cleave glycosidic linkages within polysaccharide chains, would bind more than one monosaccharide unit flanking both sides of the glycosidic bond to be hydrolysed. Crystal structures of the E4 cellulase, for example, reveal six monosaccharide-binding subsites. These sTaken together, these results are highly suggestive of a convergently evolved glycoside hydrolase catalytic site lying in the large, conserved inter-domain cleft. By analogy with the cellulase catalytic mechanism it can bBacteroides thetaiotamicron GxGYxYP-encoding genes into PULs. These loci are particularly powerful manifestations of the genome context approach to function prediction since proximity data are backed up by extensive transcriptomic profiling[Various lines of evidence indicate the general involvement of the GxGYxYP family in carbohydrate metabolism, most compellingly the grouping of all four rofiling.The first family structure reported here allows a more specific function prediction for the GxGYxYP family to be made, namely glycoside hydrolase activity. This derives first from a strong prediction of the location of a functional site, with both conservation and geometric analyses pointing to a cleft lying between an N-terminal domain of unusual, internally repeated structure and a C-terminal barrel fold. Within the cleft three conserved acidic residues, each predicted to have a perturbed pKa value as commonly seen for catalytic residues, lie in Predicting a precise substrate will likely require further experimental data. BT2193, whose structure was determined, resides in a PUL of unknown carbohydrate specificity. Two other GxGYxYP proteins lie in a PUL responsive to α-mannans, used experimentally to determine loci involved in degradation of mannose-rich cores of host N-glycans. The PUL, extending from BT3853 to BT3862, contains two other GH enzymes, one (BT3958) from family GH92, which was shown to function as an α-1,3-specific mannosidase. TogetheCaldithrix abyssi genome too. This bacterium of uncertain classification was isolated from a deep-sea hydrothermal chimney sample and its limited characterisation[Interestingly, genes for mannosidases of GH92 and GH38 families lie near those encoding GxGYxYP proteins in the risation and unpuWe provide strong evidence from a wide variety of bioinformatics techniques that the GxGYxYP family, highly over-represented in gut bacteria, constitutes a new class of Glycoside Hydrolases. Further work will be required to determined substrate profiles for members and thereby to explain the strong association with the gut microbiome.B. thetaiotaomicron VPI-5482 (ATCC No. 29148D-5) was obtained from the American Type Culture Collection (ATCC). Protein production and crystallization of BT2193 gene product was carried out by standard JCSG protocols[http://smb.slac.stanford.edu/jcsg/QC). Atomic coordinates and experimental structure factors to 1.25 Å resolution (PDB code: 3sgg) have been deposited in the Protein Data Bank .GxGYxYP family members were obtained by an iterative jackhmmer database search,73 with http://www.pymol.org/), which was also used for structure figures.DALI was usedThe authors declare that they have no competing interests.QX refined the structure. QX and YC reported on the crystallography. RYE carried out the domain and genome context analyses. RYE and DJR did the phylogenetic analysis. DJR did the structure-based function prediction. AG analysed over-representation in MetaHIT. RYE, DJR, AG and HJG interpreted and synthesised the results. All authors wrote the paper and approved the final draft.Data collection and refinement statistics (PDB code 3sgg).Click here for fileSequence alignment of selected GxGYxYP family proteins. Identical residues are shown on a red background, conserved residues are shown in red in open boxes. The secondary structure is shown above the alignment. The alignment was displayed using ESPript.Click here for file


Protein kinase C (PKC) is comprised of a family of signal-regulated enzymes that play pleiotropic roles in the control of many physiological and pathological responses. PKC isoforms are traditionally viewed as allosterically activated enzymes that are recruited to membranes by growth factor receptor-generated lipid cofactors. An inherent assumption of this conventional model of PKC isoform activation is that PKCs act exclusively at membrane-delimited substrates and that PKC catalytic activity is an inherent property of each enzyme that is not altered by the activation process. This traditional model of PKC activation does not adequately explain the many well-documented actions of PKC enzymes in mitochondrial, nuclear, and cardiac sarcomeric  subcellular compartments. Recent studies address this dilemma by identifying stimulus-specific differences in the mechanisms for PKC isoform activation during growth factor activation versus oxidative stress. This review discusses a number of non-canonical redox-triggered mechanisms that can alter the catalytic properties and subcellular compartmentation patterns of PKC enzymes. While some redox-activated mechanisms act at structural determinants that are common to all PKCs, the redox-dependent mechanism for PKCδ activation requires Src-dependent tyrosine phosphorylation of a unique phosphorylation motif on this enzyme and is isoform specific. Since oxidative stress contributes to pathogenesis of a wide range of clinical disorders, these stimulus-specific differences in the controls and consequences of PKC activation have important implications for the design and evaluation of PKC-targeted therapeutics. Figure 1). PLC exists as a family of enzymes with highly conserved catalytic domains that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the calcium-mobilizing second messenger molecule inositol trisphosphate (IP3) and diacylglycerol (DAG), the lipid cofactor that allosterically activates PKC. PLC enzymes are subdivided into subclasses based upon the distinctive structural features in their regulatory domains and their diverse roles in cellular responses. PLCβ isoforms are activated by heterotrimeric G protein subunits (Gαq and Gβγ) and mediate G protein-coupled receptor (GPCR) responses. In contrast, PLCγ isoforms are recruited to activated receptor tyrosine kinases (RTKs) where they are activated as a result of tyrosine phosphorylation by receptor or non-receptor tyrosine kinases. Since many GPCRs transactivate RTKs, PLC-γ isoforms have been implicated in both RTK and GPCR signaling responses.Protein kinase C (PKC) comprises a multigene family of related serine/threonine kinases that mediate a vast number of cellular signaling responses. Cardiomyocytes co-express multiple PKC isoforms that have been implicated in responses as diverse as cardiac contraction, cardiac hypertrophy, cardiac fibrosis, ischemia/reperfusion injury, and ischemic preconditioning , 2012. PConventional models of PKC activation focus exclusively on the second messenger-driven translocation mechanisms that deliver these enzymes in an active conformation to their membrane-delimited substrates. An inherent assumption of this model is that PKC isoforms act as generic enzymes with little-to-no substrate specificity. However, studies over the past several years have exposed additional mechanisms that alter the cofactor-requirements and/or catalytic properties of individual PKCs. Among these, the redox-dependent mechanisms that regulate PKCs are particularly interesting, since oxidative stress is a prominent feature of most clinically important cardiac disorders. While there is ample evidence that increased levels of reactive oxygen species (ROS) result in adverse cardiac remodeling and deleterious changes in contractile performance through direct redox-dependent modifications of calcium regulatory proteins and/or structural proteins in the sarcomere, this review focuses on redox-activated mechanisms that directly or indirectly influence signaling by individual PKCs. These constitute alternative mechanisms to influence pump function and/or the pathogenesis of heart failure that could provide strategies for the development of novel therapeutics.Figure 2). Structural features in the regulatory domain provide the basis to subdivide PKCs into distinct subfamilies. Conventional PKC isoforms  contain two discrete membrane-targeting modules, termed C1, and C2. The C1 domain is comprised of tandem cysteine-rich C1A and C1B sequences, with a signature HX12CX2CX13-14 CX2CX4HX2CX7C motif . The six cysteine and two histidine residues in each of the twin C1 domain structures coordinate zinc ions that stabilize the zinc finger structures. C1 domains contain the binding sites for DAG and tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA). The C1 domain lipid-binding sites are surrounded by a band of hydrophobic residues that form an extended hydrophobic surface that penetrates the lipid bilayer and provides a strong driving force to anchor the C1 domains to the DAG-containing membrane. However, membrane-docking interactions based upon C1 domain binding to DAG are not sufficient to stabilize full-length cPKCs at membranes. Rather, cPKC stably associate with membranes as a result of a second membrane-targeting interaction involving the C2 domain. cPKCs C2 domains of are ∼130 residue eight-stranded anti-parallel β-sandwich structures with three inter-strand Ca2+-binding loops (CBLs) responsible for calcium-dependent anionic phospholipid binding.Protein kinase C isoforms are structurally defined by a highly conserved C-terminal catalytic domain  that is separated by a flexible hinge region from the N-terminal regulatory domain . While nPKC C1 domains function as lipid-binding membrane-targeting modules (much like their counterparts in cPKCs), nPKCs are maximally activated by agonists that promote DAG accumulation or by PMA, without a calcium requirement. This difference can be attributed to a specific sequence difference between cPKC and nPKC C2 domain. nPKC C2 domains lack the critical calcium-coordinating aspartic acid residues that are highly conserved in cPKCs. As a result, the PKCε C2 domain does not bind calcium; it is a membrane-targeting module that binds lipids in a calcium-independent manner. The PKCδ C2 domain is even more distinct from a functional standpoint, as it does not bind lipids. Rather it is a protein–protein interaction domain that binds phospho-tyrosine residues flanked by the consensus sequence (Y/F)-(S/A)-(V/I)-pY-(Q/R)-X-(Y/F)  also have twin C1 domains and a C2 domain, although the ordering of nPKC isoform C1 and C2 domains, along the linear sequence of the protein, is switched relative to the order in cPKC (-X-(Y/F) . Recent -X-(Y/F) . The genRegulatory regions of both cPKC and nPKC also contain a pseudosubstrate motif just N-terminal to their respective C1 domains. The pseudosubstrate motif sequence resembles a PKC substrate with an alanine substitution at the phosphoacceptor site (or P-site). For both cPKCs and nPKCs, an intramolecular autoinhibitory interaction between the regulatory domain peudosubstrate motif and the kinase domain substrate-binding pocket maintains a low level of basal catalytic activity.a of its thiol moiety; cysteine residues adjacent to basic amino acids (Arg or Lys), aromatic amino acids, or metal centers, have relatively low pKa’s (<6.5) and tend to be susceptible to oxidation. The resultant cysteine oxidation can lead to disulfide bond formation and conformational changes that alter protein structure and function. Since many enzymes have reactive cysteines in their catalytic centers, oxidation of these cysteine residues leads to enzyme inactivation. One important class of enzymes with extremely low pKa active-site catalytic cysteine residues is protein tyrosine phosphatases (PTPs). Since protein tyrosine phosphorylation is controlled through the reciprocal actions protein tyrosine kinases (PTKs) and PTPs – and PTPs are extremely sensitive to redox-inactivation – this tips the balance in favor of increased protein tyrosine phosphorylation. This explains PTPs strategic position as a mediator of oxidant stress-dependent signaling responses.Cells generate ROS through a variety of tightly controlled metabolic and cellular processes that derive from mitochondrial respiration and/or growth factor stimulation. High levels of ROS also are produced during ischemic injury or in response to many pathologic insults. There is growing evidence that the subcellular location or the chemical nature of the reactive species produced in a particular setting determine whether ROS function as second messengers in signal transduction pathways or as agents for cellular injury. A major mechanism for ROS-induced cellular regulation involves chemical modification of reactive thiols in cysteine (or to a lesser extent methionine) residues in proteins. The reactivity of a particular cysteine residue is determined by the pK1-adrenergic, or endothelin-1 receptors) activate signaling pathways that lead to increased ROS generation; the receptor-dependent increase in ROS in turn activates molecular targets that play critical roles in both physiologic and pathologic growth responses . For example, H2O2 treatment increases epithelial growth factor receptor (EFGR) phosphorylation and oxidation at Cys797, a critical residue in the active site of the kinase domain  2 domains to phospho-tyrosine residues on activated (tyrosine-phosphorylated) RTKs. Receptor-anchored PLCγ is then tyrosine phosphorylated at critical sites that are essential to increase PLCγ activity. PLC-β isoforms do not have SH2 domains, are not regulated through tyrosine phosphorylation, and are not redox-sensitive enzymes. A number of laboratories have demonstrated that oxidative stress leads to a functionally important increase in PLCγ tyrosine phosphorylation . Studies527 in c-Src), which releases this autoinhibitory constraint; the enzyme then autophosphorylates at a positive regulatory site in the activation loop of the catalytic domain (Tyr418 in c-Src). The Src SH2 domain contains cysteine residues with pKa’s low enough to be oxidized by physiological levels of ROS. Direct oxidative modification of these SH2 domain cysteine residues leads to the formation of intra-molecular disulphide bridges that secondarily disrupt the autoinhibitory Tyr527-SH2 domain interaction, leading to enzyme activation (277 in Src) also has been implicated as a target for oxidative modifications that underlie redox-dependent changes in Src kinase activity  also are activated through a number of redox-driven mechanisms. Src kinases are structurally characterized by regulatory SH3 and SH2 domains and a catalytic domain, that terminates in a C-terminal regulatory sequence containing an autoinhibitory tyrosine residue; Src kinases are maintained in a closed inactive conformation with low levels of basal enzyme activity as a result of an intramolecular autoinhibitory SH2 domain-C-terminal phospho-tyrosine interaction . Src kintivation . Src alsactivity . Of noteactivity . Src kinThere is limited evidence that free radicals can directly oxidize membrane phospholipids; PLC-dependent cleavage of phospholipid hydroperoxides results in the formation of diacylglycerol hydroperoxide which acts as a very potent stimulator of PKC in inflammatory neutrophils . The rel2+-release channel or SR Ca2+-ATPase (SERCA). RyRs are macromolecular complexes comprised of four RyR protein monomers (each with a molecular mass of ∼565 kDa) bound to channel stabilizing calstabin (or FK506 binding protein) subunits and various regulatory kinases, phosphatases, and phosphodiesterases  that partitions to the nucleus and acts as an unregulated/mislocalized enzyme to stimulate gene programs that promote pathological structural and functional cardiac remodeling . Of noteFigure 3). The cysteine residues in the regulatory C1 domain are particularly sensitive to oxidative modifications that disrupt this structure and lead to kinase activation. Studies of mechanism suggest that oxidative modification of these cysteine residues (which are precisely spaced to form a hairpin structure that coordinates zinc ions) result in the release of zinc atoms and a conformational change that disrupts the C1 domain lipid-binding pocket leading to the loss of phorbol ester binding activity  may be pertinent, given the high level of sequence homology between these enzymes. In the case of PKA, Cys199 in the activation loop is a target for S-glutathionylation . Studies of PKCα suggest that this highly conserved chemical environment is functionally important, since the oxidative state promotes PKCα activation loop site dephosphorylation  studies to date indicate that these lipid interactions play a structural role to regulate actin filament assemble , this allosteric model for PKC enzyme activation by lipid cofactors has two major limitations. First, a model that focuses exclusively on lipid cofactor-mediated translocation events that deliver the enzyme to membranes assumes that the cellular actions of PKCs are exclusively membrane-delimited. In fact, PKC isoforms are recovered in mitochondria and soluble fraction of cardiomyocytes subjected to various forms of oxidative stress ; PKCs inassemble . There iassemble .Early studies showing that PKCδ is activated in a stimulus-specific manner in various cell types provided the rationale to consider a role for oxidative stress as an alternate mechanism to activate PKCδ. Specifically, studies in cardiomyocytes established that GPCR agonists drive PKCδ to lipid-membranes membranes where it phosphorylates membrane-delimited substrates. The GPCR-dependent pathway does not lead to a detectable increase in PKCδ tyrosine phosphorylation. In contrast, oxidative stress releases PKCδ from membranes, activates Src, promotes PKCδ-Src complex formation, and Src-dependent PKCδ tyrosine phosphorylation which increases PKCδ’s catalytic activity . Several52 and Tyr187) and a series of tyrosine residues in the kinase domain  that resembles the sequence flanking Tyr311 (311 is the only phosphorylation site in PKCδ flanked by residues that conform to a PKCδ-C2 domain consensus-binding motif (VGI-Y311-QGF). We used a FRET-based approach to show that PKCδ’s phosphotyrosine-binding C2 domain interacts with the Tyr311-phosphorylated V3 region . Ser357 sits at the tip of the Gly-rich ATP-positioning loop (G-loop) within the small lobe of the kinase domain. The G-loop sequence is highly conserved in most protein kinases and it forms a flexible clamp that orients the γ-phosphate of ATP for transfer to substrate; localized changes in the position of this loop influence the kinetics of nucleotide binding and catalysis. We showed that PKCδ displays a high level of S357 phosphorylation in resting cells and that the redox-induced docking interaction between the C2 domain and the Tyr311-phosphorylated hinge region leads to S357 dephosphorylation.A mechanism that might explain how a phosphorylation at Tyrg Tyr311 . Tyr311 Mutagenesis studies implicated G-loop dephosphorylation as a mechanism to regulate two major aspects of PKCδ’s enzymology . First, 23/Ser24, but it does not phosphorylate Thr phosphorylation sites on cTnI or cTnT. The observation that the redox-activated (Tyr311-phosphorylated/Ser357 dephosphorylated) form of PKCδ acquires activity toward threonine phosphorylation sites on cTnI and cTnT provided the rationale to test whether G loop phosphorylation constitutes a mechanism to regulate PKCδ’s P-site specificity. By using a series of rigorous biochemical approaches, we were able to show that the G loop phosphorylation site – which  occupies a tight space adjacent to the P-site on substrates – regulates P-site specificity. We showed that a non-phosphorylatable S357A substitution facilitates (and a phosphomimetic S357E substitution completely abrogates) PKCδ’s Thr kinase activity. Activity toward substrates with Ser-phosphoacceptor sites is not affected. These results implicate G loop Ser357 phosphorylation as a novel dynamically regulated mechanism that controls PKCδ’s P-site specificity. It is worth emphasizing that a post-translational modification within the kinase domain that calibrates an enzyme’s P-site specificity in a stimulus-specific manner is unprecedented. It is tempting to speculate that the stimulus-specific differences in PKCδ phosphorylation profiles (that impact on substrate specificity) underlie PKCδ’s diverse roles in both the pathogenesis of ischemia-reperfusion injury and as a mediator of ischemic preconditioning  leads to a conformational change that exposes bipartite nuclear localization signal in the catalytic domain. This results in an interaction with importin-α, which is key for the nuclear translocation of full-length PKCδ and various PKCδ-driven proapoptotic responses. Oxidative stress also undoubtedly increases PKCδ phosphorylation at other tyrosine residues throughout the enzyme, but the controls and consequences of other redox-induced tyrosine phosphorylations  have not been interrogated. This remains fertile ground for future investigations.Finally, it is worth mentioning that other tyrosine residues in the regulatory region of PKCδ, that figure prominently in the transduction of genotoxic stress responses, can also become phosphorylated in the setting of oxidative stress . For exaThis review summarizes our current understanding of various redox-activated mechanisms that regulate signaling by PKCs. While this narrative considers individual mechanisms separately, PKCs are subject to an elaborate and complex assortment of redox-driven modifications in various pathophysiologic settings. An understanding of the ensemble effects of these redox-driven events presents both a challenge and an opportunity for the development of novel PKC-targeted therapeutics.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Overall, we observed a rich and diverse set of spontaneously produced analogical comparisons. The high degree of expertise within the observed group along with the richly comparative nature of the economics discipline likely contributed to this analogical abundance.The use of analogy is an important component of human cognition. The type of analogy we produce and communicate depends heavily on a number of factors, such as the setting, the level of domain expertise present, and the speaker's goal or intent. In this observational study, we recorded economics experts during scientific discussion and examined the categorical distance and structural depth of the analogies they produced. We also sought to characterize the purpose of the analogies that were generated. Our results supported previous conclusions about the infrequency of superficial similarity in subject-generated analogs, but also showed that distance and depth characteristics were more evenly balanced than in previous observational studies. This finding was likely due to the nature of the goals of the participants, as well as the broader nature of their expertise. An analysis of analogical purpose indicated that the generation of concrete source examples of more general target concepts was most prevalent. We also noted frequent instances of analogies intended to form visual images of source concepts. Other common purposes for analogies were the addition of colorful speech, inclusion  of a target into a source concept, or differentiation between source and target concepts. We found no association between depth and either of the other  The importance of analogy has been described in many ways by notable researchers. Polya  wrote thin vivo and in vitro studies to fully understand biological phenomena, Dunbar  behaviors are unconstrained by laboratory conditions and are not instigated by artificial or experimental stimuli, (b) the setting emphasizes processes rather than outcomes, and (c) there is a clear relationship between observed behaviors and the domain of interest , (b) regional refers to a source mapped from a similar domain , and (c) local maps a target to a source in the same domain. Both local and regional classes are collectively referred to as within-domain, or within-category. Because of the subjective nature of judging domain distance between similar domains, many observational studies simply categorize analogies using a binary choice of within-category or outside-category. Analogies formed within the same category might compare biological organisms or investment strategies, while commonly referenced domains for outside-category analogies include sports  and the supernatural .A second important feature in analogy use concerns the range, or . Dunbar  defined Research on the domain separation of analogies has provided contrasting results. Dunbar's studies of scientific reasoning set in microbiology laboratories showed heavy use of within-category analogies—98% of the analogies generated were classified as either local or regional Dunbar, , 1997. AOne explanation for the difference in domain distance is that the collective expertise of the audience may influence the selection of analogy. When addressing other domain experts with specialized knowledge, within-category analogs may prove more effective whereas an outside-domain analogy might be more attractive choice for a general audience. The type of task or function performed by the analogizer is another possible constraint. Our second goal was to observe the use of within- and outside-domain analogies produced by the economics experts. We expected to find a fairly balanced use of both styles, which differs somewhat from earlier findings. Our expectation was motivated by the different subdomains and varying levels of subject expertise within the reading group. Furthermore, in light of the stated belief that within-domain analogies tend to involve a higher degree of superficial similarity than outside-domain analogies, we investigated whether such a relationship emerged.Thirdly, past research has shown that the goal of the individual producing the analogy is likely to influence the process of selection and transfer  weekly reading group at The University of Texas at Dallas during the Spring semester of 2011. Participants included male and female reading group attendees. The average weekly attendance was approximately 20 participants, which included both faculty members (~25%) and graduate students (~75%). The group was largely the same from week to week, but attendance did fluctuate and not all participants attended regularly. Participants' academic expertise was mixed, and included sub-disciplines such as econometrics, experimental economics, and game theory. A few of the attendees had no exposure to experimental methods.The sessions were conducted in typical reading group fashion. One of the senior faculty members acted as the group's moderator, and one student participant was assigned each week to lead the following week's discussion of one or more chosen research papers. Following a brief presentation of the paper by the assigned student, a free and open discussion followed in which the members of the group examined and dissected the experimental methods and results described in the paper.The investigator attended and made audio recordings at each of five group meetings, but did not participate in the discussions and appeared to have minimal impact on group interaction. Group members understood that their discussions would be evaluated (the moderator referred to it as “the science behind the science”). The research goal of examining the spontaneous use of analogies was not revealed, however. Although each session was scheduled to last for 90 min, actual discussion times ranged from 65 to 110 min. In all, approximately 7 h of discourse were recorded.Four transcribers participated in the initial processing of the discourse—the primary author and three undergraduate Psychology students. Only the primary author and one of the undergraduates had any significant transcription experience prior to the task. None of the undergraduate transcribers had exposure to Economics beyond introductory coursework. Transcribers were given instruction by the primary author on what constitutes an analogy based on the definitions presented earlier in this paper, then solved practice problems on recognizing analogies from non-analogies by identifying sources and targets. The period of instruction lasted approximately 30 min.For purposes of indoctrination, each undergraduate transcriber was given one of the sessions for practice and directed to process at least 30 min of the recording. They were instructed to transcribe passages in which a possible analogy was made, taking care to include all of the important source and target information. Their instruction was: “when in doubt, include it.” The primary author evaluated their performance and made individual adjustments until the results were satisfactory and consistent.To address the possibility of subtle, easily-overlooked analogies, every audio recording was processed by two transcribers. To be included for analysis, a passage needed only to appear on either transcriber's log, not both. In order to be missed, a passage would have to have been heard by both transcribers and rejected.The two authors performed the coding duties. Both were experienced coders with strong knowledge of analogy literature and considerable research experience. Each of the transcribed segments was first evaluated for the presence of one or more analogies based on the definition stated earlier. Comparisons based only on literal similarities were considered non-analogies. If a segment was judged to contain no analogy, no further evaluation was performed. If an analogy was deemed present, the source, target, source domain, and target domain of the analogy were recorded. Each analogy was then coded along the three dimensions of distance, depth, and purpose.within-domain and outside-domain categories. Following the example of Saner and Schunn . See Appendix A for a sampling of extracted passages. When judging whether a passage contained an analogy, the coders agreed on 96% (n = 109) of the 114 passages. Of these, most were rated as containing an analogy . Passages lacking the basic ingredients of an analogy  or showing evidence of a literal comparison  were eliminated. Five passages were inconclusive and were likewise eliminated. The inter-rater reliability for identifying analogies was strong (κ = 0.85). Some of the passages were found to contain multiple analogies. In all, 97 analogies were extracted for the remainder of this analysis.Transcription of the five recorded sessions yielded 114 unique passages with possible analogies (n = 88) of the 97 analogies. Of these, analogies showing no obvious overlap in surface characteristics  far outnumbered those where superficial similarities were present . The inter-rater reliability was found to be substantial (κ = 0.75).In coding for analogical depth, the coders agreed on 91% (n = 83) of the 97 analogies. Of these, within-domain  and outside-domain  analogies were almost evenly distributed. The inter-rater reliability was again found to be substantial (κ = 0.71).In coding for analogical distance, the coders agreed on 86% , Inclusion (indicating that the target was a type or component of the source), Example (indicating that the target was an instance of the source concept), Visualization (intended to create a picture or image in the mind of the audience), Emotion , Critique (using the source to point out shortcomings in the target), Exaggeration (gratuitous use of colorful phrasing), and Abstraction . Where an analogy plausibly served multiple purposes, the raters chose the strongest.n = 77) of the 97 analogies. Examples were the most prevalent , followed by visualizations . A fair number of exaggerations , inclusions , and differentiations  were observed, but abstractions  and critiques  were uncommon. The inter-rater reliability for analogical purpose was substantial (κ = 0.74).In coding for analogical purpose, the coders agreed on 79%  and within-domain  analogies. The superficial analogies were likewise split between outside-domain  and within-domain . It was determined that there was no significant distance-depth association [χ2N = 75) or structural-visualization analogies . Though the distribution of purpose ratings was highly skewed, it was skewed similarly over both categories of analogical depth and no significant depth-purpose association was found [χ2N = 71). On the other hand, when an analogizing by example, a source-target transfer from within the same domain was most commonly observed .In contrast to the two findings above, an examination of analogical distance as a function of the speaker's purpose did reveal a significant association [χThe most prominent observation to come from this study was that the use of analogy for exploration and explanation among economics experts and students was rich and abundant. In 427 min of discourse, 97 analogies were extracted, suggesting that analogies are both commonly used and serve as an important component in human reasoning and in understanding problems. The reading group setting enabled the observation of actual experts spontaneously forming analogies using their semantic knowledge applied to economics, a domain likely to entail more freedom to move between and among domains of knowledge than the previously investigated biology and political domains. The selection and fluid assembly of analogies during discourse may help to reveal the core principles involved in analogical thinking among experts. This study's findings will be discussed in the context of prior evidence.We chose to study behavioral economics experts in this study. Economists seek to broadly explain complex real-world human behavior in the context of equations, models, games, and hypothetical examples. All of these techniques rely heavily upon comparisons between different states of the world, different types of behavior, and combinations of models and situations. While some feature or object-based similarity occurs in comparisons between economic models and real-world behavior, it can be argued that the majority of the comparisons are about relations and systems of relations. For example, in the economic Ultimatum Game , but within-category analogies accounted for a sizeable portion of the total as well (47%). These results fall squarely between the previous findings, but a plausible explanation can be made. If the number of within- and outside-domain analogies is a function of expertise, then the balance between them might be expected to vary with the range and depth of expertise. In the EPPS reading group, the participants all had some degree of expertise in economics, but their domain experience varied in terms of sub-discipline  and academic career longevity . Hence, common expertise is likely to be an influencing factor, as Dunbar suggested, but the amount of influence it has on source distance may moderated by the variability in such factors as range and depth. Additionally, the goals of the participants differed from those in earlier studies. Here, the participants sought to comprehend papers describing experimental methods and results, often by comparing unfamiliar methods to known, more familiar ones. To accomplish these goals, perhaps a more balanced and diverse set of analogies is most effective.Another possibility for explaining the balance between within- and outside-domain analogies that we observed is concerns the economics discipline itself. In Dunbar's , 1997 biIn terms of a possible relationship between distance and depth, it has been suggested that within-domain analogies present more superficial similarity than distant analogies; in other words, the greater the distance, the less superficial the comparison  and non-discovery  activities. The primary difference in purpose between the economics reading group and groups observed in the cited studies was the absence of discovery goals in the reading group. Since we determined that the discussions we observed were largely non-discovery in nature, we focused on categorizing the extracted analogies into groupings based on the perceived reason for selecting the particular analogy; e.g., to differentiate a concept from other concepts, to inject emotion or colorful language into a comparison, to give a concrete example of a more abstract idea, etc. The list we derived can be found in the Results section above.It is fairly well established that goals influence the production of analogies Dunbar, . Prior sBlanchette and Dunbar  providedIn interpreting our results, we should mention that the categories we developed were not mutually exclusive; e.g., an analogy intended to differentiate between concepts might do so using visual elements. In cases where an analogy plausibly served multiple purposes, the raters chose the category they felt best described its purpose.We have already emphasized that the collected passages contained a wealth of analogies rich in depth and structure, many of which involved implied systems of complex relations that could not be fully identified and analyzed. Furthermore, some of the more complex comparisons actually involved multiple analogies at different distances and depths. The challenges we faced in terms of analyzing the passages were made more difficult due to the complexities of the structural and superficial analogies used. Unlike laboratory tasks, in which comparisons between explicit statements are made . Second, the rating process involved some inherent subjectivity; even though inter-rater reliability was rather high, there were disagreements on specific passages. This is a challenge inherent in real-world analogical analyses, as the dynamic and unscripted nature of the interactions can produce text that is very difficult to interpret outside of the context in which it was spoken and by an individual who does not have access to the speaker's intent. Third, the rating process was further complicated when analogies were embedded in familiar language constructs—well-known clichés, common metaphors, etc.—and were not immediately identified as analogies by the raters, as such phrases have become so conventionalized that their figurative qualities can be simply overlooked.We were able to ascertain some of the major purposes for which analogies are used in economics discussions among experts. The leading purpose for analogical comparisons was to provide examples. Good examples from other domains or familiar sources can provide clarity to a target and make the concept more concrete. In many of the analogies, we observed that experts tended to either relate an abstracted theoretical model to a more common situation that occurs in human interactions, or they did the reverse and described a particular situation as being an example of what is known to occur within a particular theoretical game. Experts also made common use of analogies to create a visual image of a particular concept. Visualization is important for providing a common ground for the audience and for making a target domain richer and easier to comprehend. Experts also used analogies in order to add color or interest to their contributions and to mark a topic as being included within a more familiar source domain. Given that we conducted this observation process in an academic setting, our experts may have developed tendencies to use analogy due to their experiences with teaching, in which good examples and visual comparisons can be useful for conveying concepts (Richland et al., In addition to these questions, several limitations with regard to the method and analysis of this study should be noted. The transcribers were non-experts in economics, but they were chosen to avoid domain bias and reduce the tendency to add their own interpretation to the passages. Additionally, three of the four transcribers lacked prior transcription experience in recognizing analogies, which opened the door for subtle analogies to be overlooked. We addressed this limitation using a two-phased training process and a two-person extraction strategy and believe that we reduced - but may not have eliminated - the likelihood of missed analogies. While it is possible that some subtle analogies may have been overlooked, we believe the number to be small enough to not alter our findings.Their lack of domain expertise, however, may have contributed to the subjectivity and inconsistency mentioned above. Furthermore, we did not code the analogies according to any sort of standard domain taxonomy, as Dunbar  did in hIn summary, this study reinforces the strong reliance humans place on analogies for developing understanding and communicating in natural settings. It contributes valuable evidence that humans are quite agile in their selection of analogies, drawing on a mix of shallow and deep comparisons and determining an effective distance strategy based on the constraints of the domain and the level of perceived group expertise. Economics faculty and graduate student experts engaged in scientific discussion were observed to apply analogies that were more balanced in terms of categorical distance and structural depth than those observed in other natural settings. The domain context, problem-solving goals, and participant expertise of this particular group setting all appear to be important factors that led to differences in the magnitudes of depth and distance observed in the earlier studies. No evidence of an association between the distance and depth characteristics was found, but distance and purpose appeared to be related. A number of other questions remain to be answered about the ways in which analogies are applied in different types of settings. Future naturalistic analogy research could help to clarify the role on analogical comparisons in real-world settings and the social sciences appear to provide particularly rich domains for additional studies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology  very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis  Dystroglycan (DG) is composed of two subunits, α and β, that are formed from a unique precursor which undergoes a proteolytic event within the endoplasmic reticulum during the first steps of its maturation pathway , 2. DG rDG has been particularly studied within skeletal muscle, a tissue where its α-subunit is found to be highly glycosylated, since a number of neuromuscular diseases involving glycosyltransferases active in muscle have been identified during the last years . The comThe α-subunit plays multiple important roles in DG, in that i) it is crucially involved in the maturation and posttranslational modification of the precursor; ii) it is highly modified with sugar chains especially in its central region (i.e. the mucin-like portion) from where some essential sugar moieties protrude to bind laminin. The first is based on the noncovalent binding activity of α-DG toward β -DG  while foT. termophilus , that is thought to be crucial for extracellular matrix protein binding, to O-mannosylated α-DG [The exact molecular mechanism leading to hypoglycosylation of α-DG carrying the T190M (T192M in human) mutation and causing a severe dystrophic phenotype, accompanied by some degree of intellectual impairment, is currently unknown . Via co-ted α-DG . TherefoKanagawa and colleagues have shown that the N-terminal domain of α-DG is shed by furin, in a process that is likely to take place into the intracellular Golgi compartments in which the final steps of the DG maturation process are completed . An alteO-glycans decorations along the endoplasmic reticulum and modifications accomplished in the Golgi, are crucial for its ability to function as an extracellular matrix (EM) receptor and namely to bind to EM proteins such as laminin, perlecan, agrin and neurexin in the brain and pikachurin in the retina [Interestingly, following the findings of a recent computational docking study, it was hypothesized that the T190M/T192M mutation might affect directly the binding of α-DG to laminins . Howevere retina . Notablye retina . Recentle retina ,42.Although a few glycosylation sites have been proposed and/or localized within the N-terminal and C-terminal domains of α-DG, most of the O-linked oligosaccharide chains protrude from Thr and Ser residues placed within its central mucin-like domain ,43,44. AUnraveling the molecular mechanism of disease induced by T190M/T192M is likely to be rather complex and additional hypothesis can be formulated. For example, the unveiled structural basis for the recognition of an O-glycan, GalNAc-Neu5Ac, and its attached mucin-like peptide GPATPAP by the paired Ig-like type 2 receptor α  suggestsPossibly, in solution, the effect of the T190M mutation, which is next to the flexible hinge connecting the two subdomains, may “propagate” to the Ig-like domain by changing its relative orientation with respect to the S6-like domain also influencing its binding properties. It is worthy of note that the presence of a second Ig-like domain within the C-terminal region of α-DG has been confirmed by bioinformatics analysis and molecular modeling; the domain has been biochemically characterized and shown to include the β-DG binding epitope .Such interesting structural hypotheses necessarily await further rounds of experimental/computational validations by means of techniques being able to explore the dynamical behavior of α-DG . Furthermore, molecular modeling and docking tools will be exploited to investigate the likelihood of α-DG binding to LARGE or to other regulatory factors important for DG maturation. The present structural analysis of the N-terminal T190M variant of α-DG may pave the road to the elucidation of the molecular mechanism of a specific case of dystroglycanopathy, with important consequences for clinicians and for designing possible therapies, but in addition it may be also relevant from a cellular and molecular biology perspective, helping to improve the overall picture of the multiple structural aspects underlying the maturation and function of the DG complex.


Whilst ∼93% of domain superfamilies appear to be relatively structurally and functionally conserved based on the available data from the CATH-Gene3D domain classification resource, the remainder are much more diverse. In this review, we consider how domains in some of the most ubiquitous and promiscuous superfamilies have evolved, in particular the plasticity in their functional sites and surfaces which expands the repertoire of molecules they interact with and actions performed on them. To what extent can we identify a core function for these superfamilies which would allow us to develop a ‘domain grammar of function’ whereby a protein's biological role can be proposed from its constituent domains? Clearly the first step is to understand the extent to which these components vary and how changes in their molecular make-up modifies function. Current Opinion in Genetics & Development 2015, 35:40–49Genomes and evolutionThis review comes from a themed issue on Antonis Rokas and Pamela S SoltisEdited by Issue and the EditorialFor a complete overview see the Available online 3rd November 2015http://dx.doi.org/10.1016/j.gde.2015.09.005http://creativecommons.org/licenses/by/4.0/).0959-437X/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (Families of proteins arise through speciation (orthologous relatives) and through duplication of genes during evolution  and it is the paralogues that are most likely to diverge, although not necessarily . By clasMany resources now exist for classifying protein families, some of which consider the entire protein  dramatic changes in catalytic machinery occur . HoweverHowever, dramatic changes in chemistry can occur, such as in the Enolase superfamily , 42, AldInterestingly, in some enzyme superfamilies, functional families with significantly different catalytic machineries have highly similar functions and substrates, suggesting either convergence within the superfamily or evolutionary drift from a common functional ancestor along different routes, that is, perhaps a trajectory to a less efficient enzyme with subsequent mutations restoring the activity or even resulting in a more efficient form of the enzyme. It is difficult to distinguish these cases without robust phylogenetic analyses. Such studies on Rubisco, an abundant protein important for carbon fixation, show that a more efficient form of Rubisco has emerged by convergent evolution more than 62 times in harsh environments, and structure-based analyses reveal mutations in the active site loop and secondary shell, where they possibly influence rearrangements of the active site; also at interfaces in the oligomer suggesting a role in allostery •.Enzyme superfamilies showing the greatest versatility in CATH-Gene3D, frequently adopt alpha/beta structures, two thirds having TIM or Rossmann folds. As Tawfik and his colleagues have reported in a recent publication, these structures tend to have regular, well-packed structural cores and the catalytic residues mainly locate to loops largely detached from these cores and therefore perhaps better able to tolerate the destabilising effects of mutations , 50••.Diversity in protein superfamilies can also arise from mutations in protein interfaces. Furthermore, relatives can exploit completely different surfaces in their protein interactions. Large-scale studies comparing CATH-Gene3D functional families showed that in 645 highly versatile superfamilies, cumulative binding sites from diverse relatives covered most of the protein surface and were associated with a wide range of protein partners •. HoweveAlthough only 10% of the CATH-Gene3D functional families have structural representatives, this data can help identify superfamilies capable of great structural plasticity where relatives display considerable diversity due to extensive residue insertions and repetitions or inserted structural motifs , 57. ForDramatic structural rearrangements can also arise from variations in repeating units. In the Vicinal Oxygen Chelate (VOC) superfamily, members share a common βαβββ subdomain that is organized into different topological (or domain-swapped) combinations in different relatives that maximizes the catalytic versatility of the metal center . These aDiversity can also emerge from changes in less structured regions, for example, repeats giving rise to low-complexity regions (LCRs), such as polyalanine or polyglutamine runs. These often evolve rapidly and can have a major influence on the transcriptional activity of the protein . SimilarGene fusions are another evolutionary mechanism conferring diversity as they can significantly alter the context of a domain  of the protein), thereby modifying its molecular function and biological role. Domains have been frequently duplicated and shuffled within genomes, during evolution, with fusions being more frequent and generally occurring at N or C termini . For 92%However, changes in domain partners can also alter specificity. For example, in the highly diverse Thiamine pyrophosphate (TPP)-dependant enzyme superfamily changes in domain partnership alter the size and physicochemical properties of the active site pocket (see Diversity within a superfamily can also be the result of individual relatives having multiple functions. For example, relatives can have multiple catalytic activities not necessarily of equal efficiency, as in promiscuous enzymes; or moonlighting functions whereby proteins perform completely different functions to their native activity sometimes involving different sites , 69. ProAn increasing number of proteins are now known to moonlight and these activities can be induced by oligomerisation, cellular localization, differential expression and substrate concentration. For example, Albaflavenone monooxygenase in the Cytochrome P450 superfamily, also functions as a Terpene synthase, an activity not observed in any other superfamily member. The catalytic machineries for the two enzymatic reactions are located in distinct pockets on the domain and the reactions are carried out at different pHs .In most large diverse superfamilies, functional diversity results from a combination of different molecular mechanisms . For exaExperimental data on functional diversity grows slowly as detailed studies are time-consuming and expensive, however, classifying the millions of sequences accumulating in public repositories like UniProt into putative functional families can reveal subtle changes in conservation patterns that suggest shifts in binding specificities or catalytic machineries. These data can guide experiments to focus on unusual relatives and more comprehensively landscape the functional repertoires of the most versatile superfamilies. For example, sequence similarity networks based on protein families can help in providing a comprehensive summary of sequence, structure and function relationships in a functionally diverse superfamily. Recent studies , 76 of sOnly ∼63% of the 25 million domain sequences in CATH-Gene3D can be assigned to an experimentally annotated functional family and less than 10% of these families have a known structure, so there may be much more diversity to discover. Certainly analyses of microbial communities hint at exciting novel chemistries , 78. Alt• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as: