Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur ; ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs  were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 , 71 , 22  and 4 ; vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation.Using a complete set of RING domains from  Almost all eukaryotic organisms possess numerous RING proteins. E3 ubiquitin-ligase (E3) activity is intrinsic to RING domains of c-Cbl, AO7, and seven other randomly selected RING proteins, and is likely to be a general function of the domain. Numerous RING proteins are likely to belong to RING-containing domain ubiquitin-ligases (RING-E3s) Arabidopsis thalianaPreviously, genome-wide functional analysis of RING proteins have been performed in the human and Drosophila melanogaster and all the solved RING domains from primary to tertiary structure, compared domain architecture and subcellular localization of RING proteins of Drosophila melanogaster, mapped interolog interactions of RING-E3 and E2 pairs, and pinpointed the intermolecular interface features of 3D complexes of E2/RING-E3 pairs, and the results showed that:Rapid advances in evolutionary genomics and structural bioinformatics, together with the availability of an ever-growing number of genome sequences and previously solved three-dimensional (3D) structures of RING domains, it is now possible for us to extract sequence, structural, and functional information from the evolutionary history of RING protein superfamily by comparative and structural genomic approaches. To better understand the molecular basis and principally responsible factors for the similarity and diversity of RING-E3 functions and E2/RING-E3 interactions, we comprehensively analyzed a complete set of RING domains of Drosophila melanogaster were identified and classified based on the shared sequence patterns of the conserved Cys/His residues.By comparative genomic approaches, a complete set of 139 nonredundant RING proteins from Drosophila melanogaster by Reciprocal Best Blast Hits Based on the notions: a) one-on-one mapping of protein functionality across species is a critical component of comparative genomic analysis Analyzing sequence and structural elements based on multiple alignments indicated that the large majority of RING domains have a similar second structural arrangement of ββα motif, which appears to be highly efficient for structural stabilization of RING domains.Drosophila melanogaster and the human, 117 pairs  were found to retain entirely uniform domain architectures, only Diap2/CG8293 of Drosophila melanogaster IAP family experienced evolutionary expansion of domain architecture. Additional domain analysis showed that several zinc-binding domains  widespread occurred in these RING proteins.Of the 118 orthologous pairs from Data from all the solved RING domains showed that 4 regions  are responsible for the homologous folding of RING domains at the superfamily level across long evolutionary periods, and belong to evolutionary structurally conserved regions (SCRs) .Using sequence consensus levels and conservation indices, we defined consensus and conservation of 4 hydrophobic residues and 2 polar residues located at the SCRs of RING domains. The 4 SCRs function in the appropriate positioning of the 6 equivalent residues as structural determinants for RING-E3 catalysis.Surveying spatial distribution of residues in RING domain 3D structures showed that the 4 hydrophobic residues promote the formation of the conserved core of solvent inaccessibility and consolidate within a large hydrophobic patch flanked by the 2 polar residues.Subcellular localization analysis showed that members of RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 , 71 , 22  and 4 . Of C3HC4 type, 34 members (34/68 = 50%) are located nucleus, while 21 of C3H2C3 (21/29 = 72.4%) are located multiple subcellular compartments.Drosophila melanogaster ubiquitination system.Mapping interolog interactions of E2/RING-E3 pairs showed that CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s in Analyzing all the solved and modeled 3D complexes indicated that their intermolecular interfaces form conserved hydrophobic contacts (CHCs) with E2s. Residues of RING domains directly interacting with E2s are all from the SCRs of RING domains. Of the 6 residues in RING domains, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core of the solvent inaccessibility, and 2 hydrophobic residues and 2 polar residues directly participate in E2/RING-E3 interactions by hydrophobic and electrostatic interactions.Drosophila melanogaster, the study offered a new perspective for better understanding molecular link between structural conservation and diversification, and a functional similarity and specificity of RING domains, and E2/RING-E3 interactions involved in RING-E3 catalysis.By analyzing RING domains across primary through tertiary structures, pinpointing the intermolecular interface features of 3D complexes, comparing domain architecture and detecting subcellular localization of RING proteins from Drosophila melanogaster were identified with extensive database searches and followed by manual curation to remove truncated and/or redundant sequences. Based on the shared sequence conserved patterns of the corresponding site residue-binding zinc ions, RING proteins identified here were subdivided into eight types: C3HC4, C3H2C3 (RING-H2), C3HC3D, C4HC3 (RINGv), C3HGC3 (RING-G), C4C4 (RING-C2), C6H3C2D, and U-box  detected in Arabidopsis thaliana could not be identified in Drosophila melanogasterAsp substitution at a metal ligand position, but their metal ligand positions are different (the former at position 5 and the latter at position 8). Members of the C3HGC3 type have a Gly substitution at metal ligand position 5. One or 2 members  with substitutions at a different metal ligand position  without the putative orthologs from the human independently occurred in C3HC4 (9), C3H2C3 (4), C4HC3 (3) and C3HGC3 (5) of Drosophila melanogaster. With less alternatively spliced transcript, the 21 RING proteins encode 25 mRNA of RING proteins, of which 16 lack themselves alternative splicing isoforms. As basic unit of genes, and footprints of origin, exons in genes are highly suitable for studying origin and evolution of genes Drosophila melanogaster, we performed BLAST search using each exon of the 21 RING proteins against Drosophila melanogaster genome reference sequence, and also compared the annotation of Transportable Elements available in RepeatMasker. Two long interspersed element (LINE), and one DNA element were identified to be involved in the exonization of the 21 RING proteins of Drosophila melanogaster  were found to be putative orthologs from the human , S1. Witnogaster . Withoutnogaster . TherefoDrosophila melanogaster along with a large number of orthologs between the human and Saccharomyces cerevisiae offer direct evidence in favor of the notion that RING proteins have experienced strong selective pressure for conservation throughout eukaryotic evolution Drosophila melanogaster (7). Because the record of NP_689741 was removed as a result of standard genome annotation processing in the current version, there are 8 RING/U-box domain proteins in the human, while there were 9 RING/U-box domain proteins in the previous genome annotation. Human NP_001121684 is provided with a typical RING/U-box domain ; however, its putative ortholog (NP_649969) from Drosophila melanogaster lacks the corresponding domain, resulting in RING/U-box proteins from Drosophila melanogaster having one less than the human  of 49 in Saccharomyces cerevisiaeDrosophila melanogaster and the percent distributions are similar to that of the human. While bioinformatic analysis has implicated that Arabidopsis thaliana C3H2C3-type RING domains  represent the largest, followed by the C3HC4 type Drosophila melanogaster, 6 members of C3HGC3 type all lack themselves alternative splicing isoforms, and only one (NP_648919) could be defined as its putative ortholog (NP_060594) in the human , the values are comparable with the average percentage identity of the solved RING-E3s (Cys/His) in RING domains are conserved; the values of sequence identity are actually low. If we calculated percent identity of sequence pairs by merging all alignments (10) into a comprehensive multiple alignments of 196 RING domain sequences, the percent sequence identity would be very low. Some of them may possess almost undetectable sequence identity but still converge on a common tertiary structure of the RING domain.Subsequently, we collected a nonredundant set of the solved 57 RING domains with experimental structural data, and performed multiple sequence and structural alignments for different type RING domains of nogaster  and the nogaster . Alignmenogaster . A totalnogaster . The aveRING-E3s . Owing tDrosophila melanogaster without experimental structural data were predicted by the Advanced Protein Secondary Structure Prediction (APSSP) program, which indicated that most members have a similar second structural arrangement of the ββα motif (data not shown). Of the solved RING domains, except 1BORA and 2CSZA, all others have a similar secondary structural arrangement of the ββα motif. Antiparallel β-strands are linked by a short loop of 2 to 5 residues, one of which is frequently Gly/Pro, which may be attributed to the fact that they both can assume the unusual dihedral-angle conformation required for a tight turn. The central helix connecting the first and second coordination sites of the zinc ions varies in size from several to some ten residues. Considering the obvious differences of secondary structure between 1BORA, 2CSZA, and others, we further analyzed their secondary structures using the nearest neighbor and neural network approach by the APSSP program, indicating that they both have a similar secondary structural arrangement of ββα motif to others (data not shown). Therefore, the secondary structural differences between 1BORA, 2CSZA, and others may result from the experimental conditions used when solving their 3D structures.The second structural arrangements within different types were represented according to structural data of the solved RING domains . Second Cys/His residues, members from the C3HC3D and C6H3C2D/C types have been shown to form a third zinc ion-binding site. Most of them have an Asp substitution at metal ligand positions 8 or 12 instead of Cys residues  and RING/U-box, the 8 metal-chelating residues are respectively located at the N-loop, short loop between antiparallel β-strands, α-helix, and C-loop. The positions and properties of the 4th and the 5th metal-chelating residues in these members are changeable depending on the types of RING domains. With a number of additional residues . BecauseOrthologous proteins are expected to retain function more often than other homologs.Drosophila melanogaster and the human, and the results showed that: 1) of the 21 Drosophila melanogaster RING proteins without putative orthologs from the human, 15 have the only one RING domain. Considering the data of exonization of Transportable elements, exon duplication and exonization of intronic sequences, we proposed that exonization of intronic sequences contributed mainly to the origin of the RING proteins without putative orthologs from the human after the divergence time of 700 million years ago (Ma) for the vertebrates-arthropods split As basic functional units, conserved domain architecture are required for proteins to perform their conserved function. Ari-2/Triad1 contains a N-terminal and a C-terminal RING domains , both ofDrosophila melanogaster IAP family contains 4 members . Of them, two (CG12284 and CG8293) containing RING domain possess E3 activity. Their substrates include molecules involved in apoptosis and signaling, and function in apoptotic and nonapoptotic processes. As building blocks of protein structure, domains can be utilized to recombine in different arrangements to create proteins with different functions. To assess the evolutionary dynamics behind domain architecture of orthologous Iap2/Diap2 from the human, we obtained all the possible orthologs of Iap2/Diap2 from distant phylogenetic lineages, and performed phylogenetic analysis for these orthologous Iap2/Diap2 (Caenorhabditis elegans) and fungi (Schizosaccharomyces pombe and Magnaporthe oryzae) are organized by a tandem repeat of 2 BIR domains. Without RING domain, the orthologs from nematode and fungi were omitted in our analysis; 2) all orthologous Iap2/Diap2 from arthropods possess a tandem repeat of 3 BIR domains and 1 RING domain; 3) apart from a tandem repeat of 3 BIR domains and 1 RING domain, all orthologous Iap2/Diap2 from vertebrates acquired an additional Card domain after the divergence time of 700 Ma for the vertebrates-arthropods split Drosophila melanogaster molecules Reaper, Grim, Hid, Sickle, and the caspase Dronc Drosophila melanogaster and the human, and the occurrence of large numbers of orthologs between the human and Drosophila melanogaster, and the low sequence similarity based on pairwise sequence comparison implicated an ancient common origin of the 118 putative orthologous pairs, and also emphasized the notion that RING proteins have experienced strong selective pressure for conservation throughout eukaryotic evolution.p2/Diap2 , S4. DomDrosophila melanogaster  do not contain any other detectable, previously described domain. However, majority of genomic proteins, more than 80% in metazoa, are multiple domain proteins As structural basis and potential source of functional diversity of RING proteins, additional domains outside RING domain should play important roles in functional diversity, substrate specificity and usability of general RING protein function. Carp2 (NP_001017368), a negative regulator of TNF-induced NF-kappaB activation, by virtue of its phospholipid-binding FYVE/RING domain, Carp-2 localized to endocytic vesicles, where it interacted with internalized TNF-receptor complex, resulting in RIP ubiquitination and degradation Cys/His residues are filled in a more orderly fashion than the rest of the RING domains in spite of the noncorrelation of sequences between some of them. To better understand evolutionary-variable and -conserved regions of the RING domains, we analyzed whole and local main-chain conformation using all the solved 3D structures of the RING domains. As a straightforward methodology for detecting structural similarity, pairwise RMSD values were first calculated by the RMSD metric to evaluate their whole structure similarity. With reliable RMSD values (average ≤3.0 Å) for pairwise C-alpha atom superposed by sequence alignments, most of these structures possessed general similarity  are unlikely to be a general feature of its superfamily. In other words, the Cys/His residues involved in the stability of RING domain structure are the most conserved, which ensures RING domain folding more conservation than its residues' sequences. To infer to what extent consensus and conservation of additional amino acids occurs but not the conserved Cys/His residues, we used the above alignments which revealed that 6 residues near the conserved Cys/His pairs in the SCRs of the N-loop, βα-region and C-loop have a higher degree of consensus and conservation than others in RING domains. These agree well with residues of conservation in RING Domains from Arabidopsis thalianaIle (located at the N-loop SCRs) in the C3HC3D, C3HGC3, and C6H3C2D types; the second hydrophobic residues of Phe (located at the βα-region SCRs) in the C3HGC3 and C6H3C2D types; and the third hydrophobic residues of Ile (located at the βα-region SCRs) in the C3HGC3 and U-box types are completely conserved. And the fourth hydrophobic residues, located at the C-loop SCRs, are Pro in all members from the C3H2C3, C3HC3D, C4C4, C3HGC3, C6H3C2D, and U-box types. The first polar residues, located at the α-helix of the βα-region SCRs, completely differ from each other. They are in positively charged residues such as Lys and Arg in some members, while replaced by negatively charged residues such as Glu or Asp in others. The second polar residues, located at the C-loop SCRs, are primarily occupied by Arg. Evidently, RING/U-box and RING/non-U-box types not only share consensus and conservation of residues but the residues in the U-box are also located at the proximity of those that stabilize the RING/U-box domain  were merged into a comprehensive multiple alignment of 196 RING domains. A preliminary comparison of all superfamily members showed that additional residues  at the α-helix has also been shown to directly participate in the intermolecular interfaces for Cont4 and UbcH5B (1UR6) by electrostatic interactions In addition, most members from C3HC4, C3H2C3, C3HC3D, C4HC3, C4C4, and C6H3C2D types have one Main-chain conformation and sequence profile highlight the importance of the 4 SCRs and 6 equivalent residues. Do these observations truly reflect common structural constraints found within the core of RING domains at the tertiary structure level during evolution? Are the 4 SCRs and the consensus and conservation of residues in RING domains the critical signal, or just noise for RING domain structure and function? To address these questions, using all the solved RING domains, we first analyzed and compared spatial distribution of the 6 equivalent residues at the SCRs by mapping them onto their 3D structures. The calculation of spatial distance between residues indicated that the hydrophobic residues were clustered in space in close proximity by centering around the second hydrophobic residues at the second β-sheet of the SCRs. Specifically, the minimum spatial distance between any one of them and the second hydrophobic residues is less than 4 Å. Analysis of spatial distribution of the equivalent residues in 3D structures demonstrated that the hydrophobic residues (located at the N- and C-loops), distant from each other in their primary and secondary structures, can finally achieve spatial proximity in 3D structures and convene within a large hydrophobic patch, which is flanked by the 2 polar residues of consensus and conservation.Cys/His, the four hydrophobic residues mediate a conserved hydrophobic core packing of solvent inaccessibility. By expulsing water, the four hydrophobic residues are key to the formation of the conserved core of solvent inaccessibility, which appears to be an inherent properties of RING domain 3D structures  surface for all hydrophobic residues of the solved RING domains based on the VDW radius of each atom in the molecule, colored by their electrostatic potential . Substitution of the internal residue Phe25 in Rhodobacter sphaeroides thioredoxin by 5 amino acids , of which the aliphatic amino acid substitutions  significantly decreased the protein stability that was possible due to loss of extensive VDW contacts that Phe25 made with its neighboring residues. The F25Y (Tyr) substitution did not evidently affect protein stability, which may be attributed to the similar property of the Tyr residue to Phe in possessing a large bulky aromatic side chain, that can adopt similar VDW contacts with its neighboring residues as Phe25 does Tyr residues at the positions of central hydrophobic residues in the solved RING/U-box domains  are occupied by a large bulky aromatic  domains . By VDW bic core , which mDrosophila melanogaster, the fourth hydrophobic residues at the C-loop in the solved RING domains were mainly occupied by Pro residues, accounting for 50/57 (87.7%). By fixing the main chain dihedral angle at approximately −65±11°, the rigid pyrrolidine ring of Pro may play an important role in hydrophobic contact of intermolecular interfaces of RING-E3 and E2 complexes. The second polar residues at the C-loop in the solved RING domains are also overrepresented by the positively charged Arg residue (24/57 = 42.1%), which may profit from the following Arg residue properties: 1) the largest side chains of the guanidino group attached to the residue contributes significantly to the formation of complexes of E2/RING-E3 by increasing surface contact from a distance; 2) flanking the hydrophobic patch, the positively charged Arg residue may contribute to the stability of 3D complexes by electrostatic interactions.Considering the fact that cavities on a protein surface create the physicochemical properties needed for a protein to perform its function, using the solved RING domain 3D structures, we analyzed surface accessible pockets, as well as interior inaccessible cavities by CASTp Drosophila melanogaster were found to be located ≥2 subcellular compartments. As a membrane-associated RING-E3 located multiple subcellular compartments, RNF11 negatively regulate NF-kappaB and jun N-terminal kinase signaling pathways Gly2 and the S-palmitoylation of Cys4. RNF11 mutated in the palmitoylation signal is retained in compartments of the early secretory pathway Eukaryotic cells are elaborately subdivided into functionally distinct membrane bound compartments. Protein localization tends to be tightly bound to its function, and represents the primary functional information of RING proteins. Subcellular localization analysis of these RING proteins by WoLF PSORT Drosophila melanogaster and the human are evolutionary conserved, which may suggest they have a common core function. Experimental function data of orthologous RING proteins showed that, of 118 orthologous RING proteins, 88 RING-E3 activities have been confirmed by experimental data . And CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than others in Drosophila melanogaster ubiquitination system. Topors functions not only in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UBE2D1-3 and UBE2E1 for p53 Mapping human E2/E3-RING interactions have provided us a detailed, genome-wide overview of binary E2/E3-RING interactions in human ubiquitination system nogaster . Data anIle383 and Trp408 of c-Cbl and the UbcH7 Phe63 have a central role in determining the specificity of the c-Cbl E3 for the E2 Phe63 is Asn65, and the residue corresponding to the c-Cbl Trp408 is His55 Phe63 of UbcH7 and Trp408 and Ile383 of c-Cbl are replaced by Ser70, Gln59, and Ser84 respectively To illustrate how an E2 can accommodate different RING-E3s, previously, experimental analysis of key residues of RING-E3 have been conducted in Cbl, cIAP2, Traf6, Cont4, El5, Rad5p, Vmw110, Sh3rf1 and Chip RING-E3s . DespiteDrosophila melanogaster were respectively constructed by homology modeling approaches; 2) using the RMSD metric, we measured the mean distance between the corresponding atoms in the 2 structures after targets and templates were superimposed by sequence alignment using C-alpha in each residue; 3) with quite reliable RMSD values (0.05 to 0.984), we obtained high-quality modeled complexes of E2/RING-E3 pairs  is a bulky positively charged Lys (K370), and is replaced by the small hydrophobic residue Pro (P71) for the RING domain from Traf6 (3HCTA); 3) in the α-helix of the αβ-region SCRs, most complexes include residues 1, 2, and 3 that directly participate in E2/RING-E3 interactions. Of the 3 residues, the polar residues demonstrate consensus and conservation in the above alignments , replaced by a polar residue of His in Chip (2OXQC: H241). These observations are taken together with the following 2 facts: a) the positions and properties of the 4th and fifth metal-chelating residues in the SCRs are changeable depending to some extent on the types of RING domains; b) RMSD values of the βα-region SCRs showed more difference than others  and 1UR6B (20 residues) . And thipro, ile, val) from the SCRs. Of the 6 consensus and conservation of residues, residues 2 and 3, as structural conserved residues, contribute to constructing the hydrophobic conserved core of solvent inaccessibility in the CHCs. Residues 1 and 5 directly participate in interactions within the CHCs of intermolecular dynamic surfaces.  from the RING/U-box domain of CHIP produces electrostatic interaction with the positively charged Lys of position 5 (K5) UBE2N (2C2VT) Clearly, the intermolecular interfaces of 3D complexes exhibit conserved and diversified features. From an evolutionary perspective, conservation and consensus of residues may be essential for mediating the formation and maintaining the stability of the hydrophobic conserved core of solvent inaccessibility, and offering the CHCs flat for RING-E3s and E2s recognizing and binding underlying catalysis, while diversification of individual residues of their dynamic interface should be necessary for selective targeting to different E2s. From a structural perspective, the invariant equivalent positions of interacting residues across different RING-E3s suggests that: 1) the integrity and the precise relative arrangement of conserved residues and structure are important for RING-E3 function; 2) despite poor conservation of interaction residues in the various complexes of E2/RING-E3 pairs, their roles in direct structural or functional effects on E2s or potentially allowing conformational changes during catalysis by hydrophobic and electrostatic interactions or hydrogen bonds are common; 3) variable sequences of RING domains likely determine their selective binding properties because each RING domain interacts with a subset of available E2 proteins; the importance of individual residues in RING-E3s can vary from each other depending on different E2/RING-E3 pairs DrosophilaThe present investigation was facilitated by the readily availability of the solved RING domains, the cocrystal structural complexes of E2/RING-E3 pairs and genome sequences, and the findings of previous studies Drosophila melanogaster were comprehensively retrieved from GenBank (http://www.ncbi.nlm.nih.gov/), Uniprot (http://www.uniprot.org/), Supfamily (http://supfam.cs.bris.ac.uk/), InterPro (http://www.ebi.ac.uk/Tools/) and FlyBase (http://flybase.org/) databases. Under the default profile inclusion expectation (e) value threshold, profile searches were conducted using the Position-Specific Iterated BLAST (PSI-BLAST) program with either single RING domain sequence or multiple alignments as queries, which were iterated until convergence. Under the default expected threshold parameters, several online tools, including the Simple Modular Architecture Research Tool 6 (http://smart.embl-heidelberg.de/), CD-search (http://www.ncbi.nlm.nih.gov/), the ScanProsite Proteomics Server (www.expasy.org), the Protein Families database (Pfam 24.0), and the European Bioinformatics Institute (http://www.ebi.ac), were used to analyze and confirm each potential RING domain, and proteins containing RING domains were retained for further investigation. Putative orthologs of RING proteins between Drosophila melanogaster and the human were further defined by Reciprocal Best Blast Hits (RBBHs) Drosophila melanogaster were clustered and subdivided into eight types. Multiple amino acid sequence alignments of these types were performed by the Tcoffee-regular program under Expresso (3DCoffee) computation mode cys/his residue positions. Simultaneity and confidence level of sequence alignments were evaluated using structural information by Expresso and Promals3D, which construct alignments using structural information from sequence database searches, secondary structure prediction, available homologs with 3D structures, and user-defined constraints.RING proteins from http://www.wwpdb.org/), until no new sequences were found under the e-value threshold. To identify additional RING proteins of the solved 3D structures with similar topology, structure similarity searches were conducted by DaliLite (version 3.0), which uses Heuristic filters to rapidly find a close neighbor of the queried structure Using either single RING domain sequence or multiple alignments as queries, an initial search for all nonredundant members of RING domains with experimental structural data were iteratively carried out with the PSI-BLAST in PDB database  and ExPASy InterPro Scan Drosophila melanogaster and the human were evaluated. All the possible orthologs of Iap2/Diap2 were obtained from distant phylogenetic lineages. Amino acid of all these orthologous Iap2/Diap2 were further analyzed by multi-sequence alignment using Expresso, which automatically incorporate structural information in multiple sequence alignments using 3D-Coffee Domain architecture of RING proteins of the 118 putative orthologous pairs from Homo sapiens, Build 37.2), chimpanzee , Sumatran orangutan , rhesus , mouse , rat , cow , dog , cattle , horse , rabbit , duck-billed platypus , opossum  chicken , zebrafish , western clawed frog , fruit fly , honey bee , jewel wasp , red flour beetle , nematode , fission yeast , rice blast fungus . In additional, WormBase, FlyBase, VectorBase, SGD, DictyBase, M. brevicollis, v1.0 and Gramene Homepages were also retrieved by basic local alignment search tool (BLAST) (see reference database).The investigated species include human (w = 3) position was then scrolled through the alignment and used to define seed positions with a mean RMSD≤3.0 Å in JavaScript. Once a seed position was found, w was iteratively increased by one position consecutively until the mean score did not rise above 3.0 Å, or until the window reached the end of the multiple alignments. In addition, the solved RING domain 3D structures were used to calculate the fraction of hydrophobic residues it encapsulates along a sequence by Scooby-Domain http://sts.bioengr.uic.edu/castp/) were also used in the data analysis.A central tenet of structural biology is that related proteins of common function share structural similarity. To identify the SCRs of RING domains, we first inspected the superposability of the solved RING domain under a threshold of RMSD http://swissmodel.expasy.org/), whose 4 tools  were utilized. In addition, the PDB database was searched for template identification. For the computational simulation of 3D complexes of RING-E3s docking to E2s from Drosophila melanogaster, we first obtained all the cocrystal structures of RING-E3 and E2 pairs with experimental structure data, including 6 cocrystal structures  Drosophila melanogaster by RBBHs. Sequence and structural alignments were conducted as described above; once an accurate alignment was determined, 3D complex models of orthologous RING-E3 and E2 pairs from Drosophila melanogaster were generated with Alignment Mode by computational simulation of coordinate templates. In addition, the solved RING-E3s (2EA6A) from the human was selected as a template for modeling the corresponding orthologs (Mura) from Drosophila melanogaster. Using the cocrystal structure of 3HCT data, we performed molecular docking of Mura-Ben. Ultimately, seven 3D complexes of RING-E3 and E2 pairs, including Cbl-UbcD10, Iap2-Eff, Traf6-Ben, Cont4- Eff, Chip-Eff, Chip-Ben, and Mura-Ben were established by replacement of the coordinates of their respective templates based on their structural similarity Drosophila melanogaster were submitted to the PMDB protein model database http://accelrys.com/). The scripts show ligands and receptor binding site atoms within 4.0 Å between appropriate atoms in 3D structures for deducing the hydrophobic and polar interactions; ligand interacting residues (close approach) distance cut-off values are calculated within 3.5 Å by finding residues close to the current selection. The inferred interacting residues were further confirmed via manual examination using the Swiss-PDB viewer The constructions of 3D models were performed through the dedicated server SWISS-MODEL workspace ; C: C3HC3D; D: C4HC3 (RINGv); E: C3HGC3 (RING-G); F: C4C4 (RING-C2); G: C6H3C2D; H: U-box). The first lines of second structural arrangements of the types were respectively represented by the corresponding type orthologs with experimental structural data . The second structural arrangements of C3HGC3-type are the results of prediction by APSSP program due to lack the corresponding orthologous RING domain with experimental structural data. Green cylinders represent α-helices, green arrows represent β-strands, and grey lines represent loops. Except for U-box type without the full complement of Zn2+-binding ligands, the others are provided with the conserved Cys/His pattern. And the conserved metal ligand position and residues involved in coordinating Zinc ions were shadowed by grey. Equivalent residues of the conserved Cys/His in several members were replaced by non-Cys/His residues, which were shadowed by yellow for easy identification. Based on the previous structural evidence from 2BAY Cys/His, while C3HC3D and C6H3C2D/C types are stabilized by three Zinc ions. Because of far from RING domain, and more variability of metal ligand position and Zinc ions coordinating amino acid pairs, the third Zinc ion of C3HC3D-type was not represented. Residues  contributing to the stabilization of U-box were shadowed by grey. Gly/Pro residues in short loop between β hairpin were indicated by bold letters for easy identification. The last two lines in different types showed consensus amino acid sequence (Con) for positions with a consensus level equal to or above 0.8 and conservation indices (Cons) for positions with a conservation index above 4. Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic : l; aromatic : @; hydrophobic : h; alcohol : o; polar residues : p; tiny : t; small : s; bulky residues : b; positively charged : +; negatively charged : −; charged : c.(PDF)Click here for additional data file.Figure S2Exonization of RING proteins of fruit fly without orthologs from the human. (A) Exon duplication of CG31053 and CG12200 exon 1; (B) Exonization of long interspersed element (LINE) in CG5071 exon 1 and CG4325 exon 2; (C) Exonization of DNA element in CG17721 exon 1. The dots represent the same nucleotides as the consensus sequence. ID%: the percentage identity for pairwise sequence comparison.(PDF)Click here for additional data file.Figure S3Multiple sequence and structure alignments of the solved RING domains.A: C3HC4-type, C3H2C3-type (RING-H2), C3HC3D-type, C4HC3-type (RINGv) and C4C4-type; B: C6H3C2D-type/C6H2C4-type and U-box. Name of sequences were indicated by PDB IDs. Secondary structures of the sequences were colored according to experimentally-determined structural data . Four structurally conserved regions (SCRs)  were indicated by green rectangles. Except from 1BORA and 2CSZA, all the others have a similar second structural arrangement of the ββα motif. The top lines of the alignments indicate the consensus secondary structure (SS); Conserved Cys/His residues binding zinc ions were shadowed by grey. Consensus amino acids were showed by pansy for easy identification. One exception is 3I2D with one occurrence of zinc ion at 3, 5, 7 and 8 positions, its corresponding site residues do not bind atom of zinc were shadowed by yellow . Gly/Pro residues in short loop between β hairpin were indicated by bold letters for easy identification. The 4th metal-chelating residue position and zinc ion coordinating amino acids tend to be changeable in distinct type RING domains. The last 2 lines in different types showed consensus amino acid sequence (Consensus_aa) and conservation indices for positions with a conservation index above 4. Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic : l; aromatic : @; hydrophobic : h; alcohol : o; polar residues : p; tiny : t; small : s; bulky residues : b; positively charged : +; negatively charged : −; charged : c.(PDF)Click here for additional data file.Figure S4Sequence alignment of orthologous Diap2. All the orthologous Iap2/Diap2 from arthropods possess a tandem repeat of 3 BIR domains and 1 RING domain. Apart from a tandem repeat of 3 BIR domains and 1 RING domain, all orthologous Iap2/Diap2 from vertebrates acquired an additional CARD domain. Iap2/Diap2 from arthropods lack the corresponding CARD domain, which were indicated by broken line rectangle. Hom, Homo sapiens; Pan, Pan troglodytes; Mac, Macaca mulatta; Pon, Pongo abelii; Mus, Mus musculus; Rat, Rattus norvegicus; Orn, Ornithorhynchus anatinus; Mon, Monodelphis domestica; Bos, Bos taurus; Equ, Equus caballus; Can, Canis lupus familiaris; Ory, Oryctolagus cuniculus; Dan, Danio rerio; Gal, Gallus gallus; Xen, Xenopus (Silurana) tropicalis; Dro, Drosophila melanogaster; Drp, Drosophila pseudoobscura; Tri, Tribolium castaneum; Api, Apis mellifera; Nas, Nasonia vitripennis.(PDF)Click here for additional data file.Figure S5Analysis of main-chain conformation of the solved RING domains. (A) Superimposition of all the solved RING/non-U-box domains by sequence alignments using c-Alpha in each residue. Four regions  with reliable RMSD (average≤2.0 Å), lacking insertions and deletions were detected. (B) N-terminal loop superposed by 9 residues. (C) The first β-sheet region superposed by 7 residues. (D) βα-region superposed by 13 residues. (E) C-terminal loop superposed by 6 residues. (F) Superimposition of all the solved RING/U-box domains by sequence alingments. (G) Superimposition of 1FBVA (RING/non-U-box type) and 2OXQ (RING/U-box type) domains by sequence alingments. 2OXQ (RING/U-box type) are provided with structural extension at C-terminal . N: amino terminal; C: carboxyl terminal; β-sheet: antiparallel β-strands. The backbones of RING domains were superposed by C-Alpha atom in each residue.(PDF)Click here for additional data file.Figure S6Cartoons of VDW surfaces created for all hydrophobic residues of RING domains. Based on the van der Waals radius (VDW) of each atom in the molecule, VDW surface were created for all hydrophobic residues of RING domains, which were colored by Electrostatic potential . The spatial distribution of consensus and conservation of residues in RING domains were respectively indicated by blue (hydrophobic residues) and yellow letters (polar residues). 3D structures of RING domains were displayed by atom of ball and stick, which were colored by elements.(PDF)Click here for additional data file.Figure S7Cartoons of 3D complexes of RING-E3 and E2 pairs.A: Cbl-UbcD10; B: Iap2-Eff; C: Mura-Ben; D: Traf6-Eff; E: Cont4-Eff; F: Chip-Eff; G: Chip-Ben. A-I to G-I: Targets and templates were respectively represented by the purple ribbon models and the light blue schematic models. Structural similarity between them was shown by superimposition of their structures . Cantact interfaces of RING-E3 and E2 complexes were illuminated by purple dot circles. I and II represent the first and the second Zn-binding sites. The 2 sites are respectively located on 2 sides of RING-E3 domain, by which form a characteristic “cross-brace” zinc-binding topology of RING domains. A-II to G-II: Close-up view of interaction residues in the intermolecular interfaces of their 3D complexes. The side chains of E2 and RING-E3 3D complexes involved in E2/E3 interactions were presented by solid ribbon. Resides that make significant directly contacts observed in the modeling complexes were presented by stick model, and were numbered by precursor peptides. The numbers for all resides in the figure correspond to those in the text and the tables. The interaction residues in the interface were respectively indicated by yellow (E2s) and white (RING-E3s) letters. The conserved hydrophobic contacts of intermolecular interfaces observed in the modeling complexes were highlighted by red dot circles. Hydrogen bonds of intermolecular interfaces of complexes formed by carbonyl-group oxygen and amino-group hydrogen were showed by white dot lines.(PDF)Click here for additional data file.Figure S8Close-up view of interaction residues in the intermolecular interfaces of 3D complexes of RING-E3 and E2 pairs.A: Cbl-UbcD10; B: Iap2-Eff; C: Mura-Ben; D: Traf6-Eff; E: Cont4-Eff; F: Chip-Eff; G: Chip-Ben. The side chains of 3D complexes of RING-E3 and E2 pairs involved in their interactions were presented by solid ribbon. Resides that make significant directly contacts observed in the modeling complexes were presented by stick model, and were numbered by precursor peptides. The numbers for all resides in the figure correspond to those in the text and the tables. The interaction residues in the intermolecular interface were respectively indicated by yellow (E2s) and white (RING-E3s) letters. The conserved hydrophobic contacts of intermolecular interfaces observed in the modeling complexes were highlighted by red dot circles. Hydrogen bonds of intermolecular interfaces formed by carbonyl-group oxygen and amino-group hydrogen were showed by white dot lines.(PDF)Click here for additional data file.Table S1Drosophila melanogaster and its corresponding putative orthologs from human.Information related to RING proteins from (XLS)Click here for additional data file.Table S2Identity and similarity percentages for all sequence pairs within different types.(XLS)Click here for additional data file.Table S3Drosophila melanogaster and the human.Domain architecture comparison of orthologous RING proteins from (XLS)Click here for additional data file.Table S4RMSD values for pairwise C-Alpha atom superposed by sequence alignments.(XLS)Click here for additional data file.Table S5RMSD values for the 4 SCRs for pairwise superposed by sequence alignments using c-Alpha in each residue.(XLS)Click here for additional data file.Table S6Hydrophobic residues identified close to the central hydrophobic residues within 4 Å.(DOC)Click here for additional data file.Table S7Drosophila melanogaster and the human.Information relevant to interolog interactions of RING-E3 and E2 pairs from (XLS)Click here for additional data file.Table S8Summary of interolog interactions of E2s and RING-E3s from fruit fly.(PDF)Click here for additional data file.


The BHP allows discrimination of lactation-specific BLV prevalence within a dairy herd, to help identify risk factors and management plans that may be important in transmission of BLV.Enzootic bovine leukosis is a contagious disease of cattle caused by the retrovirus, bovine leukemia virus (BLV) and is the most common cause of malignant neoplasm in cattle. In order to facilitate surveillance of this disease in dairy herds, we developed a method to combine ELISA of milk collected during routine production testing with a prescribed sampling of cows that is independent of the proportion of cows within each lactation. In 113 Michigan dairy herds, milk samples from ten cows in each of the 1st, 2nd, 3rd, and ≥4th lactations were analyzed for anti-Bovine Leukemia Virus (BLV) antibodies by milk ELISA. For each herd, a BLV herd profile (BHP) was calculated as the simple average of the percent of BLV-positive cows within each of the four lactation groups. The mean BHP for all herds was 32.8%, with means of 18.5, 28.8, 39.2, and 44.8% of 1st, 2nd, 3rd, and ≥4th lactation animals infected, respectively. In eight herds, we determined the correlation between the BHP, and true herd prevalence by testing the entire lactating herd ( Enzootic bovine leukosis is a contagious disease of cattle induced by the Δ-retrovirus, bovine leukemia virus (BLV). The disease complex is characterized by a persistent lymphocytosis which can culminate in B-cell lymphoma. Although BLV-induced lymphoma is the most common neoplastic disease of cattle, most infected cows do not display outward signs of disease, and these animals are referred to as asymptomatic or aleukemic . ApproxiInfection of BLV, as detected by serology, is more typical of older cattle as opposed to younger cattle , 6, and Dairy herds in Michigan, that routinely participated in Dairy Herd Improvement Association (DHIA) testing, and averaged ≥120 cows on test for the previous 12 months, were stratified into equal-sized cohorts of 119 small-sized herds (120–174 cows), 119 medium-sized herds (175–295 cows), and 119 large herds . Within each of these strata, herds were assigned a random number which determined the order in which they were contacted and invited to participate in our study. Forty herds were sought from each cohort, but exclusions because of the inability to schedule herd visits, or lack of data within herd DHIA records, resulted in a total of 113 participating herds. The mean (±SEM) number of cows on test for each herd was 407.7 ± 67.0, and ranged from 113 to 6,492. 105 herds milked Holstein cows only, 2 herds Brown Swiss, 4 herds Jerseys, and 2 herds milked a mixture of breeds. In order to estimate the number of cows to be sampled per herd, we calculated, by binomial proportion of standards, that if the true within-herd prevalence was 10%, there would be a 5% probability that no positive cows would be among a sample of 28 cows, that is, the probability was 95% that we would find at least one positive cow. We selected the theoretical BLV prevalence of 10% for our calculations because this is well below the mean herd prevalence reported in several studies [n = 2, 158) to calculate BHP (based on 40 cows). The BHP was then compared to the average of lactation-specific prevalence (from whole-herd testing) and total herd prevalence by simple correlation analysis.For each of the 113 herds, the percent of BLV-positive cows was computed for each lactation group and the percentages from all four lactation groups were averaged  to calculate the BHP. In order to validate the accuracy of the BHP sampling relative to total herd BLV prevalence, we stratified all 113 herds by BHP result in quartiles  and randomly selected two herds from each quartile for further BLV testing. In these 8 herds, milk samples were collected from all lactating cows for BLV ELISA . Samples were transported to a DHIA laboratory  and subsequently analyzed for antibodies to BLV as described below . All transportation and storage of samples was at ambient temperature. All analyses for antibodies to BLV were conducted within 5 days of the original collection date. We previously determined that the stability of anti-Mycobacterium avium subsp. paratuberculosis antibodies in milk, stored, and detected by a commercial assay similar to the milk BLV ELISA, is consistent for 14 days at room temperature . Antibodies to BLV were detected using an ultrapure virus lysate in a commercially available antibody capture ELISA  routinely used for bulk milk analysis. Prior to analysis, individual DHIA milk samples were diluted 1 : 30 in sample diluent to reduce the effect of carryover contamination (<1%) that occurs during the DHIA sampling process. Briefly for the ELISA, antibodies to BLV in dilute milk samples bind to microtiter plate wells previously coated with virus lysate. Bound antibodies from milk are detected by reaction with horseradish-peroxidase-(HRP-) labeled monoclonal antibodies to bovine immunoglobulin (IgG). Bound HRP-labeled antibodies are detected by addition of enzyme substrate. Reaction times are standardized to the color development of positive controls (0.9 < 450 nm OD < 1.2) and stopped by the addition of 0.5 N H2SO4. Sample scores (sample OD-negative control OD) ≥0.1 were considered positive. Milk samples were immediately preserved with 0.2 mg/mL bronopol and 7.8 r). BLV prevalence rates among lactation groups were evaluated with one-way ANOVA analysis. Descriptive statistics regarding the frequency distributions, as well as all other tests described above, were done using SAS-PC . Correlations among herd measures of BLV prevalence were expressed as Pearson correlation coefficients (P < 0.0001 by one-way ANOVA) with or without the assumption of equal variance among the 4 lactation groups. For the eight herds in which all lactating cattle were sampled and tested for BLV by ELISA, the BHP was found to be highly correlated with the actual ELISA prevalence , and also highly correlated with the average lactation prevalence  with crude herd BLV prevalence.Overall, mean BLV prevalence of infection increased as the number of lactations increased, which likely arises from complex interactions among herd management, pathogen exposure, and possible limitations to detect all infected animals by routine screening with milk ELISA. The use of milk ELISA, coupled with employment of the BHP, enables a practical tool to determine age-stratified prevalence of BLV infection in dairy herds. We propose the BHP as a standard to help dairy producers manage BLV infection within their herds because the BHP (1) indicates which age groups are becoming infected, thereby suggesting which disease control interventions may be most effective, (2) provides a means to monitor the impact of disease control interventions, (3) allows comparisons with other herds, or historical comparisons within the same herd, regardless of differences in age distribution, and (4) is highly correlated (


The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes. One impp97 is a homohexamer comprising an N-terminal domain (N) and two tandem AAA domains (D1 and D2). X-ray crystallographic studies have elucidated the quaternary structure of the complex at near-atomic resolution –15 but hHere, we have used a variety of EM techniques to visualize the conformations of p97 in different nucleotide states. By separating protein conformations within the same single-particle dataset, we avoid the conformational variability of p97 that has confounded earlier analyses and are able to identify two primary conformations that exist simultaneously in solution. We show that one of these is highly dynamic throughout the nucleotide hydrolysis cycle: the position of the N domains above or in plane with the D1 ring is linked to different conformations of the D1 and D2 rings. We also measure nucleotide binding-dependent rotations of the D1 ring relative to D2. Our new reconstructions allow us to propose a mechanism that links N-domain position to conformational transitions within D1 and D2 that may be linked to the remodelling of target protein complexes.3.3.1.E578Q [Cryo-EM and single-particle analysis were used to study the conformation of p97 in different nucleotide states. To eliminate heterogeneity owing to slow ATPγS hydrolysis, a hydrolysis-deficient D2 mutant (p97E578Q ) was usea–c). The first type (side view 1) features three distinct densities at the top layer and a continuous density at the bottom layer. The second type (side view 2) features two layers of flat, continuous density, with a wider top layer. In class averages of side view projections of p97 without the N domain (p97ΔN), only side view 1 was observed (d). Together, these data suggest that differences in side view projections are owing to N-domain conformations. To confirm this, we used Ni-NTA-Nanogold to label the N-terminal His-tag of full-length p97 (e). Regardless of nucleotide state, we observed nanogold particles above  or coplanar  with the D1 ring. Occasionally, we also observed differences in the position of the gold particles within one p97 molecule . These results indicate that p97 can adopt multiple conformations and that the relative position of the N domains is a key feature of this heterogeneity.Upon examining class averages of projections representing side views of p97, we identified two major types within all three datasets a–c. The observed d. Togethngth p97 e. RegardE578Q monomer, similar to data for wild-type p97 [To investigate whether conformational changes are induced by nucleotide binding to D2, we determined the stoichiometry of prebound nucleotide in purified p97. Our previous studies showed that approximately 90% of the D1 domains of wild-type p97 contain ADP, whereas D2 is free of bound nucleotide . Using ttype p97 . Given ttype p97 , this su3.2.To separate p97 particles within the cryo-EM data, two initial models were built from the two different side views and used as references for competitive alignment within each dataset . Particles aligning to either reference were sorted into different subsets. Data processing and refinement was subsequently carried out using standard angular reconstitution procedures.a), whereas they are coplanar with the D1 ring in conformation 2 (a). The final reconstructions of conformation 1, at resolutions between 17 and 20 Å, show similar three-layered hexameric structures (figure 2a). The top layer occupies the least density and was assigned to N domains. Strongly connected density that rises in a tall dome can be seen for N domains in the apo and ADP states, whereas a flat cap is observed above the entrance to the D1 pore in the ATPγS state (figure 2a). The D1 and D2 rings form the middle and bottom layers of the reconstructions, respectively. All three structures show an expanded D1 ring with pronounced hexameric protrusions and a wide central pore. The D2 ring is narrower and shows large nucleotide-dependent changes (figure 2a), most notably in the orientation of the six D2 domains relative to the central axis of p97.Two distinct p97 conformations were identified in all three nucleotide states . In the figure 2mation 2 a. The fia). There is little conformational difference between the ATPγS and ADP structures. The N domains are coplanar with the D1 domains forming a relatively flat ND1 ring similar to what is observed in p97 crystal structures. The D2 ring is more open than that of conformation 1.Conformation 2 is primarily observed in the presence of nucleotide. Forty per cent of particles imaged in the presence of ATPγS or ADP but only about 5% of the particles in the apo dataset aligned to this conformation. Thus, only the reconstructions in conformation 2 will be described for the ADP and ATPγS states. Refinement resulted in final structures of approximately 15 Å resolution comprising a two-layered hexameric barrel with a wide top layer and narrower but more open bottom layer a. There 3.3.EM reconstructions obtained from transmission electron microscopic images have ambiguous handedness, a limitation inherent in the method. The correct handedness of all reconstructions in this work was determined by comparison with the known X-ray structure of p97. Specifically, helix 3 in the ATPase domains protrudes from the core of the domain, giving the domain the overall shape of a comma that curves clockwise (CW) when viewed from the top of p97. Clockwise curvature of the protrusions of the ATPase domains disambiguates the hand of all reconstructions . A model derived from the p97 crystal structure fits better into reconstructions of the correct hand than into those of the opposite hand.b). In addition, the D1 ring is expanded in all nucleotide states, with individual subunits rotated outward and up. The D2 ring also expands somewhat relative to the crystal structure, but this change is minor compared with that in the D1 ring.A hybrid p97 protomer  was manually placed inside the density of each of the five EM reconstructions. This initial position was then reciprocal-space refined with sixfold symmetry constraints. Fitting p97 into conformation 1 requires a large rearrangement of the N domains relative to the D1 ring. These rearrangements are modelled, in the different nucleotide states, by rotations that flip the N domain into an apical position and are constrained by the N–D1 linker b. In addb). The N domains required a slight upward rotation, but not nearly to the extent that is seen in the structure of an IMBPFD mutant of p97 [Fitting was straightforward for conformation 2, whose flat top layer shows a significant resemblance to the ND1 ring in the p97 crystal structures b. The N t of p97 . To obta3.4.The presence of nucleotide leads to a change in the conformation of the N domains. Whereas they assemble to extend the central pore in the apo structure, they occlude it in the ATPγS and ADP states of conformation 1 and are locked coplanar to the D1 ring in conformation 2 . The D1 A comparison of the relative arrangements of the D1 and D2 rings in the different nucleotide states and conformations reveals striking rotations  experiments that showed in real-time rotation of the D1 ring with respect to D2 upon the addition of ATP .In the AFM work, rotation was measured relative to the C termini of p97, which were immobilized on a mica surface. We took sections approximately halfway through the D2 ring as the point of reference. As p97 is not a straight molecule but shows distinct handedness, it is not surprising that the extent of rotation observed in AFM (23° CW) and in our work (22° CCW) differs. To ensure that our conclusions are not an artefact of a poor alignment, we also aligned our maps on their D1 domains. We still measure a rotation in the ATP-bound state of a bit more than 20° , and obtain nearly identical ring rotations for the apo and ADP states. (The direction of rotation is inverted because the motion is now relative to D1 and not to D2.) Morphs between pairs of reconstructions show that the transition to and from the ATP state goes along with a D2 domain transformation that repositions a conspicuous protrusion that we annotate as helix 3 . In summary, both our study and the earlier AFM work are in agreement that binding of ATP but not ADP induces the conformational changes in the D2 domain. Our data are furthermore consistent with the hypothesis that the location of the N-domain is coupled to inter-ring rotations upon ATP binding and hydrolysis.Because of the periodicity in a hexamer of 60°, a 22° CCW rotation and a 38° CW rotation leads to equivalent relative orientations of the D1 and D2 rings. Neither direction of rotation is implausible. On the one hand, the 22° CCW rotation appears to preserve inter-domain connectivity , an AAA ATPase related to p97 that disassembles the soluble NSF attachment protein receptor (SNARE) complex in an ATP-dependent manner, D1 rotates CCW relative to the D2 ring after γ-phosphate release . The appin vivo, either as a means of sampling conformational space or to probe the environment for interacting partners that are required for the biological function of p97. In the absence of nucleotide, conformation 1 predominates. Presence of nucleotide stabilizes conformation 2. Upon binding of ATP and repositioning of the N domain–adaptor subcomplex, ATP hydrolysis starts. The N domains are released and the D1 ring rotates back against D2 into the position observed in the apo state.It is tempting to speculate that conformation 2 represents the entrance point into ATPase cycle. In earlier experiments, when p97 was held in what amounted to conformation 2 , ATP hydrolysis was suppressed , which s5.Our structures provide global information on relative motions within the p97 hexamer that are consistent with various existing cryo-EM and crystal structures, and with recent single-molecule observations by high-speed AFM. The data indicate coordinated flexibility in the N as well as the ATPase domains, and suggest a possible way of converting the chemical energy stored in ATP into mechanical energy required for the remodelling of substrates. Given the conformational heterogeneity within p97, more detailed structural characterization by single-particle cryo-EM  will require further breakthroughs in methodology. For now, it seems clear that the biological function of p97 requires positioning of the N domain coplanar to D1 and relative rotations of the ATPase rings.6.6.1.6-tagged E578Q mutant) was expressed as described for wild-type p97 [E578Q were pooled and concentrated to 5 μM.Full-length p97  at a ratio of one p97 hexamer to one labelling particle. In samples requiring addition of ADP or ATPγS, nanogold labelling was carried out immediately after incubation with nucleotide.6.3.−1).Protein was loaded onto grids, either directly or after incubation with 100 μM of ATPγS or ADP for 30 min on ice, and attached to a Vitrobot (FEI) set to 4°C with a blotting time of 2 s. Data were collected on a Phillips CM200 FEG electron microscope using a 4 k × 4 k CCD camera (TVIPS) at 50 k magnification (1.76 Å pixel6.4.Image processing was primarily carried out in IMAGIC . Images 6.5.http://www.pymol.org). For all five reconstructions, the domains comprising the hybrid protomer  were manually and separately adjusted to obtain a good initial fit constrained by the EM densities and the relative orientation of the domains. Adjustments were as small as possible and within the reach of the N–D1 and D1–D2 linkers. These initial fits were then refined in VEDA (http://mem.ibs.fr/VEDA/) using reciprocal-space refinement with sixfold symmetry imposed.A hybrid protomer built from p97-ND1  and the 6.6.himera [Movies were created in Chimera . First,


Protein phosphorylation in eukaryotes is carried out by a large and diverse family of protein kinases, which display remarkable diversity and complexity in their modes of regulation. The complex modes of regulation have evolved as a consequence of natural selection operating on protein kinase sequences for billions of years. Here we describe how quantitative comparisons of protein kinase sequences from diverse organisms, in particular prokaryotes, have contributed to our understanding of the structural organization and evolution of allosteric regulation in the protein kinase domain. An emerging view from these studies is that regulatory diversity and complexity in the protein kinase domain evolved in a ‘modular’ fashion through elaboration of an ancient core component, which existed before the emergence of eukaryotes. The core component provided the conformational flexibility required for ATP binding and phosphoryl transfer in prokaryotic kinases, but evolved into a highly regulatable domain in eukaryotes through the addition of exaggerated structural features that facilitated tight allosteric control. Family and group-specific features are built upon the core component in eukaryotes to provide additional layers of control. We propose that ‘modularity’ and ‘conformational flexibility’ are key evolvable traits of the protein kinase domain that contributed to its extensive regulatory diversity and complexity. The catalytic core that performs this process is highly conserved and remarkably specific to substrates in signalling pathways. Since signalling pathways control important cellular processes such as transcription, cell cycle progression, differentiation and apoptosis (see reviews –4), precOver the past few years, sequence-based search procedures, crystal structures and biochemical studies have revealed several protein kinase-like (PKL) families in bacteria, archaea and in lower eukaryotes that are distantly related to the EPKs. Some of these families include aminoglycoside phosphotransferases in pathogenic bacteria , lipopolBoth EPKs and ELKs are more distantly related to several distinct classes of atypical kinases (APKs)  that speGiven the evolutionary relationship between EPKs, ELKs and APKs and the striking differences in substrate specificity and regulatory aspects of these three classes of kinases, one can ask the following important questions. First, what sequence and structural features are typical of the catalytic core of EPKs and ELKs, but not of APKs, and how do these features facilitate precise regulation of EPKs in signalling pathways? Second, what features are common to EPKs and ELKs and how do they relate to their common functions? Third, can we specifically pinpoint these features given the amount of sequence data ,32 now aHere, we describe how quantitative comparisons of the evolutionary constraints acting on EPK, ELK and APK sequences and structures have provided insights into the modular organization and evolution of regulation and substrate specificity in the protein kinase domain. We define the core structural features shared by EPKs, ELKs and APKs, and show that EPKs and ELKs have diverged from APKs through the addition of structural features that contribute to the conformational flexibility of the catalytic core. The EPK–ELK shared features are further elaborated in EPKs through the addition of flexible loops, such as the activation loop, which provides a framework for allosteric regulation by phosphorylation. We show that group and family-specific motifs within EPKs are built upon flexible regulatory segments, such as the activation loop, to provide additional layers of regulation.2.EPKs adopt the same fold as ELKs and APKs despite sharing very low sequence similarity ,33. This3.a,b). The C-lobe was also suggested to be structurally related to a distinct domain named ‘kinase non-catalytic C-lobe domain’ (KIND) based on remote homology searches -x-G motif in the F-helix tethers the activation loop and substrate-binding G-H-I helices through water-mediated and CH-π interactions (figure 4d). Why would such tethering be important for EPK functions? One possibility is that tethering provides an additional layer of regulation beyond the EPK–ELK structural component, and a framework for allosterically coupling distal regulatory sites to the active site. Consistent with this view, mutation of residues in the αC-β4 loop increase fibroblast growth factor receptor 2 (FGFR2) activity by altering C-helix and inter-lobe movement . T. TErk2),ding (a) . It is ading (a) . Likewisding (a)  and p38 ding (a) . It is i(b)b). This AGC-specific variation allows tethering of the C-tail to the αC-β4 loop by facilitating a CH-π interaction between a C-terminal tail arginine/lysine and the HxF motif phenylalanine (figure 5b). Likewise, other AGC kinase-specific motifs tether the C-tail to the N-lobe and active site of the kinase core through interactions that are specifically conserved in AGC kinases [PKA, PKB, PKC and related kinases  present yet another example of how group-specific motifs are built upon the EPK-specific features to provide additional layers of regulation. Analysis of the evolutionary constraints that most distinguish AGC kinases from other EPKs revealed that the C-terminal tail, which interacts with the catalytic core in PKA, is the most distinguishing feature of AGC kinases . Furtherfigure 5 kinases . Why wou kinases  or mutat kinases . In the  kinases . The C-t kinases ,80.From the analysis of both AGC kinases and CMGC kinases, it emerges that the kinase scaffold is quite plastic in tolerating multiple regulatory mechanisms. Moreover, each kinase seems to have evolved a unique mode of regulation by conserving motifs that bind different regulatory domains. In both the cases studied, the additional layer of regulation is mediated by insert segments or flanking sequences (CMCG insert near G-helix and the C-tail in AGC kinases). Regulation by such flanking segments has also been shown for tyrosine kinases –83, and 7.Modularity is a concept that is widely used to explain the complexity of biological systems. Indeed, the modular recombination of the catalytic domain with diverse regulatory domains has been shown to contribute to functional diversity  and


Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains  with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation. Transmission of signals across the cell membrane is an essential process for all living organisms. Integrins are one example of cell surface receptors (αβ) which, uniquely, form a bidirectional signalling pathway across the membrane. Integrins are crucial for many cellular processes and play key roles in pathological defects such as cardiovascular diseases and cancer. They are activated to a high affinity state by the intracellular protein talin in a process known as ‘inside-out activation’. Despite their importance and the existence of functional and structural data, the mechanism by which talin activates integrin remains elusive. In this study we use a multi-scale computational approach, which combines coarse-grained and atomistic molecular dynamics simulations, to suggest how the formation of the complex between the talin head domain, the cell membrane and the integrin moves the integrin equilibrium towards an active state. Our results show that conformational changes within the talin head domains optimize its interactions with the cell membrane. Upon binding to the integrin, talin facilitates rearrangement of the integrin TM region thus promoting integrin activation. This study also provides a demonstration of the strengths of a computational multi-scale approach in studies of membrane interactions and receptor conformational changes and associated proteins that enable transmembrane signaling. Integrins are cell surface receptors involved in many essential cellular processes, such as cell migration, and in pathological defects, such as thrombosis and cancer four-point-one, ezrin, radixin, moesin) domains Talin consists of a head domain (∼50 kDa) and a large rod domain (∼220 kDa) The inactive state of integrins is in part maintained by interactions between the α and β TM helices. Important interactions are defined by regions known as the outer (OMC) and inner (IMC) membrane clasps . In the complete talin head domain (i.e. domains F0–F3), a lipid bilayer, and the α/β integrin TM regions. Our results provide novel information about the orientation of the intact talin head in complex with the lipid bilayer, and about changes induced in the TM helical regions. Overall, the results reveal how binding of talin to the membrane and to integrins tails leads to integrin activation.Molecular dynamics (MD) simulations allow us to explore the conformational dynamics and lipid interactions of membrane proteins We have used a serial multiscale MD simulation tal-sol-AT) in solution were first used to explore potential internal flexibility between the four component sub-domains (F0–F3) along with possible conformations of the F1 domain insertion. For these simulations the insertion in the F1 domain (res: 134–172) was modeled in a random coil conformation, and was located away from the F0–F1 pair  The principal simulations underlying the current study are summarized in pair see  using Motal-h2F0-CG and αβ-talh2-CG) on a multi-microsecond timescale. The talin head domain/αβ complex was constructed as described in Kalli et al.αβ-talh2o-AT).The conformation of the talin head domain suggested by the above simulations was used to model the association of the talin head domain with a phospholipid bilayer. Since NMR studies suggested a helical propensity for the region involving residues 154–167 A number of control simulations were also performed to evaluate the robustness/sensitivity of the results and to explore the contributions of different regions and interactions  to the binding of the talin head to anionic lipid bilayers. Detailed descriptions of these simulations are provided in the Supporting Information  and S2. tal-sol-AT in between the F0–F1 and F2–F3 domain pairs, no significant angle change was observed within either the F0–F1 or the F2–F3 domain pair, suggesting that each pair behaves approximately as a rigid body.To study the conformational dynamics of the talin head domain prior to the association with the bilayer atomistic (AT-MD) simulations of the talin head domain (i.e. subdomains F0 to F3) in aqueous solution in the absence of a bilayer were performed  that the F1 loop interacts with F0–F1 to form a positively charged surface that extends the positive patch on F2–F3, CG simulations with the loop in this location were performed to explore the nature of the interactions of the complete talin head domain with an anionic lipid bilayer. Note that in this simulation system a small helical region  with the same POPC/POPG  bilayer showed that the positively charged region of F0–F1 augmented by the F1 loop promotes direct F0–F1/bilayer interactions (data not shown). CG models of talin with restricted flexibility between the F0–F1 and F2–F3 domain pairs did not bind to the bilayer in a way that would facilitate binding of F2–F3 to the β-tail in any of these simulations see . DisruptOverall, our simulations suggest that optimal association of talin with the membrane is enhanced by conformational flexibility within the head domain, especially between the F0–F1 and F2–F3 pairs. This flexibility facilitates optimal interactions of the V-shaped conformation of talin with the headgroups of anionic lipids. Reduction in the anionic lipid content significantly decreased the talin/bilayer association and, in the presence of mutations that disrupted the talin/lipid electrostatic interactions, talin bound to the bilayer in a perturbed orientation .tal-h2F0-CG) was modeled in a complex with the α/β integrin TM complex  with an anionic phospholipid bilayer promotes a rearrangement of the subdomains, which in turn initiates a conformational rearrangement of the integrin TM region. In particular, on binding to the membrane the F0–F3 talin head domain undergoes a conformational change to a V-shape, rather than the linear arrangement of the F0-F1-F2-F3 subdomains observed in the crystal structure. Formation of a complex between the rearranged talin head and the integrin TM domain subsequently triggers separation of the TM helices i.e. disassembly of the TM helix dimer. The first stage of this disassembly is an initial scissoring motion of the helices, as seen in our previous studies Our studies show that conformational changes in the talin head on binding to anionic phospholipid membranes mediate transmembrane signaling by the integrin TM helix dimer. Simulations have revealed how optimization of the interactions of the The current study reveals how the flexible linker between the F0–F1 and F2–F3 pairs allows talin to adopt a conformation which optimizes its contacts with anionic headgroups of lipids . This coIn silico mutations  of this surface perturb binding of talin to the membrane, in agreement with experimental studies Our simulations reveal how electrostatic interactions between the protein and anionic lipid headgroups orient the talin head domain and optimize its interactions with the membrane. The cationic surface of the talin head, which binds to the anionic lipids, is formed by the F2 and F3 domains plus the F1 loop. Experimental studies indicate that inserted loop in the F1 domain is dynamic in nature Simulations that included the entire talin head/membrane/TM complex revealed that talin interactions with the integrin β tail and the membrane surface disrupt the interactions of both the OMC and IMC regions, resulting in eventual dissociation of the TM helices. Calculation of the hydrogen bonds between the lipids and the membrane see  after thFrom a more general perspective, this study reveals the interplay of membrane interactions and conformational changes involved in transmembrane signaling by receptors and associated proteins and/or domains. In particular, it may be compared with recent simulation studies, e.g. of the EGF receptor 2. In the tal-l25-CG and tal-l50-CG simulations . In the CG simulations the center of mass of the protein was placed 120 Å from the center of mass of the preformed bilayer (2). Finally, CG-MD simulations were performed. The final snapshot of the CG-MD simulation was converted to an atomistic (AT) representation, using a fragment-based approach The CG-MD simulations were performed using a local variant ulations  the forc bilayer . This stFor the simulations with the talin/αβ complex, the same POPC/POPG bilayer as above was used. The TM region of the talin/αβ complex was inserted in the bilayer using GROMACS. The lipids that overlapped with the integrin TM region were removed. The same energy minimization and equilibration steps were performed as described above. A modified CG model was used where all the ENM restraints between the integrin α TM region and the rest of the complex were removed.www.gromacs.org) −6 bar−1 and the reference pressure was 1 bar.All CG-MD simulations were performed using GROMACS 4.5 (2). Subsequently, unrestrained AT-MD simulations were performed. All the analyses were performed using GROMACS (www.gromacs.org) The AT-MD simulations were performed using the GROMOS96 43a1 forcefield Figure S1The compete talin head domain. A. Positions of the F1 loop (in different conformations) relative to the rest of the talin head domain at the beginning of the CG-MD simulations. See (PDF)Click here for additional data file.Figure S2tal-sol-AT simulation (below). In both cases the surface shown to interact with the bilayer is oriented towards the reader. The electrostatic calculation was performed using APBS Electrostatic profile of the talin head domain. Surface electrostatic potential representation for the crystal structure of the talin head domain (above) and the final snapshot of the (PDF)Click here for additional data file.Figure S3tal-h2F0-CG simulation at 0 ns, 900 ns and 1.5 µs. The color scheme for the talin domains is the same as in tal-h2F0pc-CG with a zwitterionic (POPC) lipid bilayer. The different colored lines correspond to the five repeat simulations. The horizontal broken line indicates the distance when talin is associated with the bilayer surface.Association of the talin head domain with a lipid bilayer. A. Progress of the simulations with the complete talin head domain. Snapshots from the (PDF)Click here for additional data file.Figure S4tal-h2F0-AT simulations) between the talin F0 domain (A) and the h2 helix (B). The contacts are mapped on the F0 and h2 helix structures. Blue indicates a low number, white indicates a medium number and red a large number of contacts. Contacts are defined by using a distance cut-off of 3.5 Å between the F0 residues and the h2 helix residues.Interactions between the talin F0 subdomain and the h2 helix. A,B. Normalized average number of contacts Click here for additional data file.Figure S5tal-l4E-CG and tal-lK324D-CG simulations Click here for additional data file.Figure S6tal-AT (A) and the tal-h2F0-AT (B) simulations.Comparison of the interaction between the talin head domain and the POPC and POPG lipids. A,B. Number of hydrogen bonds between the protein and POPC (black) and POPG lipids (red) for the (PDF)Click here for additional data file.Figure S7αβ-talh2o-AT (red), the αβ-talh2p-AT (green) and the αβ-tal-AT (black) simulations. C. Number of H-bonds between the talin and the lipids (shown separated for POPC (black), POPG (red) and all (orange) lipids) as a function of time for the αβ-talh2o-AT simulation.Atomistic simulations of the talin/integrin complex in a lipid bilayer. A. Movement of the F0–F1 pair relative to the F2–F3 pair as a function of time for the three AT systems with the talin head domain. Inset pictures show the talin conformation for each case. B. The β helix tilt angle relative to the bilayer normal as a function of time for the (PDF)Click here for additional data file.Figure S8αβ-talh2o-AT simulation Click here for additional data file.Table S1Details of all talin simulations. This table provides details of all the talin simulations performed in this study.(PDF)Click here for additional data file.Table S2Details of all talin/integrin TM simulations. This table provides details of all the simulations of the talin/integrin TM complex performed in this study.(PDF)Click here for additional data file.Table S3Important residues for the talin/lipid interactions. This table provides details for all the residues that made more than 90% of the interaction with the lipids in our simulations.(PDF)Click here for additional data file.Text S1Supplementary results from control and sensitivity analysis simulations. This text provides results from a number of control simulations and simulations evaluating the sensitivity of the main results to changes in simulation setup, in particular for: (i) the relative orientation of F0–F1 and F2–F3 pairs and role of F1 loop; (ii) the role of the negatively charge lipids in talin/membrane interactions; and (iii) the talin/αβ complex.(PDF)Click here for additional data file.


The NTF2-like superfamily is a versatile group of protein domains sharing a common fold. The sequences of these domains are very diverse and they share no common sequence motif. These domains serve a range of different functions within the proteins in which they are found, including both catalytic and non-catalytic versions. Clues to the function of protein domains belonging to such a diverse superfamily can be gleaned from analysis of the proteins and organisms in which they are found.Bacteroides thetaiotaomicron (strain VPI-5482) [PDB:3KZT], Cj0202c from Campylobacter jejuni subsp. jejuni serotype O:2 (strain NCTC 11168) [PDB:3K7C], rumgna_01855) and RUMGNA_01855 from Ruminococcus gnavus (strain ATCC 29149) [PDB:4HYZ] have been solved by X-ray crystallography. All three domains are similar in structure and all belong to the NTF2-like superfamily. Although the function of these domains remains unknown at present, our analysis enables us to present a hypothesis concerning their role.Here we describe three protein domains of unknown function found mainly in bacteria: DUF3828, DUF3887 and DUF4878. Structures of representatives of each of these domains: BT_3511 from Our analysis of these three protein domains suggests a potential non-catalytic ligand-binding role. This may regulate the activities of domains with which they are combined in the same polypeptide or via operonic linkages, such as signaling domains (e.g. serine/threonine protein kinase), peptidoglycan-processing hydrolases (e.g. NlpC/P60 peptidases) or nucleic acid binding domains (e.g. Zn-ribbons). The NTF2-like superfamily is a large group of related proteins that share a common fold, first observed in the structure of the rat NTF2 (Nuclear Transport Factor 2) protein . It is aThe NTF2-like proteins can be broadly defined into two functional categories: enzymatically active and non-enzymatically active proteins. The intracellular examples of this fold include most of the enzymatic functions associated with these proteins. These include SnoaL polyketide cyclase, scytalone dehydratase, limonene-1,2-epoxide hydrolase and δ5-3-ketosteroid isomerase  and Mba1Release 27.0 of the Pfam database  includesDUF3828 family [Pfam: PF12883] is annotated in Pfam as a domain of unknown function. It is present in 492 different UniProtKB proteins from 451 different organisms. It is found exclusively in Gram-negative bacteria, with the vast majority of the species it occurs in belonging to the Enterobacteraceae family. [Pfam: PF12870] was previously annotated in Pfam as a lumazine-binding domain, however this has since been found to be incorrect and so we have renamed this family as a domain of unknown function, DUF4878. This domain is present in 650 different UniProtKB proteins from 571 different species. Like DUF3828, DUF4878 is a bacterial family, however it is found in a wider variety of bacterial species. It is found in both Gram-negative bacteria (including Proteobacteria and Bacteroidetes) and Gram-positive species (including Firmicutes and Actinobacteria). Finally, DUF3887 family [Pfam: PF13026] is another domain of unknown function. This domain is present in 364 different UniProtKB proteins from 262 different species. It is predominantly found in Firmicutes, but is also present in other phyla, including several Archaeal species.All three of these domains are of a similar length (around 100 amino acids). The N-terminus of DUF3828  containing DUF3828 also contain a DUF4878 domain at the C-terminus  . The final model includes two molecules (residues 26–167), five 1,2-ethanediol, two sulfate ions and 118 water molecules in the asymmetric unit. The structure is mainly composed of three helices, one 310 helix and 4 beta strands. Gly0 (that remained at the N-terminus after cleavage of the expression/purification tag), the region from Lys26 to Pro34 was disordered and not modeled. All the side chains were fully modeled because of the complete electron density. The Matthews coefficient (VM) is 2.05 Å3 Da-1 and the estimated solvent content is 39.97%. The Ramachandran plot produced by MolProbity  from Campylobacter jejuni subsp. jejuni serotype O:2 (strain NCTC 11168), was determined to 2.0 Å resolution by MAD method and was deposited to PDB as [PDB:3K7C]. The final model includes four molecules (residues 1–113), one chloride ion, thirteen di hydroxyethyl ether (PEG), six triethylene glycol and 134 water molecules in the asymmetric unit. The structure is mainly composed of three helices and four beta strands. Gly0 (which remained at the N-terminus after cleavage of the expression/purification tag), the region from Met1 to Ser5 was disordered and not modeled. All the side chains were fully modeled because of the complete electron density. The Matthews coefficient  is 2.3 Å3 Da-1 and the estimated solvent content is 46.46%. The Ramachandran plot produced by MolProbity shows that 97.4% of the residues are in favoured regions, with no outliers.The crystal structure of a DUF4878 protein, Cj0202c protein [UniProtKB:Q0PBT7] from Ruminococcus gnavus (strain ATCC 29149), was determined to 2.25 Å resolution by MAD method and was deposited to PDB as [PDB:4HYZ]. The final model includes two molecules (residues 36–149), six chloride ions, six sulfate ions, eight glycerol and 107 water molecules in the asymmetric unit. The structure is mainly composed of four helices, four turns, and five beta strands. Only Gly0 (that remained at the N-terminus after cleavage of the expression/purification tag) was disordered and not modeled. All the side chains were fully modeled because of the complete electron density. The Matthews coefficient (VM) is 3.00 Å3 Da-1 and the estimated solvent content is 58.98%. The Ramachandran plot produced by MolProbity shows that 99.6% of the residues are in favored regions, with no outliers.The crystal structure of a DUF3887 protein, the hypothetical protein Rumgna_01855 [UniProtKB:A7B2S7] from 03 and the structure alignment has 85 equivalent positions with an RMSD of 1.72 Å; the structures of [PDB:3KZT] and [PDB:3K7C] are significantly similar with P-value of 2.57e-03 and the structure alignment has 95 equivalent positions with an RMSD of 2.53 Å; the structures of [PDB:4HYZ] and [PDB:3K7C] are significantly similar with P-value of 1.55e-06 and the structure alignment has 95 equivalent positions with an RMSD of 2.99 Å  and [PDB:4HYZ]. FATCAT results showed that the structures of [PDB:3KZT] and [PDB:4HYZ] are significantly similar with P-value of 1.53e-f 2.99 Å . In all The three structures  all possess NTF2-like folds, despite being dissimilar in sequence  to identify related sequences in gene neighbourhoods. Each cluster of homologous proteins were then assigned an annotation based on the domain architecture or conserved shared domain which were detected using Pfam models and in-house profiles run using RPS-BLAST ; the peak(λ 1), inflection (λ 2), and high energy remote (λ 3) [PDB: 4HYZ]; or the inflection (λ 1) and high energy remote (λ 2) [PDB:3KZT], of a multi-wavelength or a two-wavelength selenium multi-wavelength anomalous diffraction (MAD). The data were integrated and scaled using the XDS and XSCALE programs respectively [Protein purification and crystallization was performed by the JCSG crystallomics core -31. All ectively ,33 [PDB:ectively  and SCALectively  programsectively  and the ectively . Iteratiectively  at a resectively  at a resectively ,41 at a ectively  and MAD-ectively  or BUSTEectively  [PDB:4HYThe quality of the crystal structure was analyzed using the JCSG Quality Control Server . This seThe data sets supporting the results of this article are included within the article . Table S3. Data collection and refinement statistics (PDB 4hyz).Data collection and refinement statistics (PDB 3kzt). Click here for file


Dobutamine stress cardiac magnetic resonance imaging (DCMR) was not an established test for coronary ischemia in 2003/2004. Meanwhile, long-term outcome has demonstrated a strong prognostic value of negative DCMR testing . However, only limited data are as yet available about reimbursement for and cost-effectiveness of DCMR in patients with stable coronary artery disease (CAD).An electronic patient information system (PIS) archive allowed the selection of a matched control group of patients undergoing cardiac catheterization (CA) without prior DCMR. Our institution takes part in the German Diagnose Related Groups (DRG) reimbursement scheme and has transmitted micro cost calculations for all in-hospital stays of patients to the InEK database since 2003. The study cohort of this "natural experiment" 2003-2004 comprises 706 patients with suspected coronary artery disease . A group of patients with DCMR guided treatment strategy (DCMR)  was matched to a control group with CA indicated by standard evaluation (SECA) (N=442).Framingham risk score did not differ significantly between groups (7.7 ± 2.8 vs. 8.1±3.2). Outcome was better in DCMR than in SECA. This trend was borderline significant in the above sample. An evaluation in a larger sample  comprising additional patients from this period without cost data demonstrated a significantly (P<0.006 log-rank test) superior outcome in DCMR that persisted even after adjustment for cardiovascular risk and revascularization. Median incremental costs related to hospital stays were 12.713 € ± 43.664 € (median 4864 €) in the SECA and 4104 € ± 4454 € (median 3387 €) in the DCMR group. Differences were located on the ward, intensive care unit and operation room but not in the cath-lab. All types of costs particularly human resource costs were elevated in the SECA group. Incremental cost-effectiveness ratio was calculated as 822 € per survival. Sensitivity analysis demonstrated that at least 750 € /per patient are saved by DCMR. Death, PCI, CABG, and hospital admissions were 6%, 1%, 1%, and 35% in the DCMR and 11%, 34%, 20%, and 97% in the SECA group over 8 years.DCMR is cost-effective in competition with SECA even if a reimbursement of 750€ for each DCMR examination is calculated. This natural experiment allows the simulation of detailed realistic cost-effectiveness scenarios for health technology assessment of DCMR.none