The Non-structural 1 (NS1) protein of avian influenza (AI) viruses is important for pathogenicity. Here, we identify a previously unrecognized tandem PDZ-ligand (TPL) domain in the extreme carboxy terminus of NS1 proteins from a subset of globally circulating AI viruses. By using protein arrays we have identified several human PDZ-cellular ligands of this novel domain, one of which is the RIL protein, a known regulator of the cellular tyrosine kinase Src. We found that the AI NS1 proteins bind and stimulate human Src tyrosine kinase, through their carboxy terminal Src homology type 3-binding (SHB) domain. The physical interaction between NS1 and Src and the ability of AI viruses to modulate the phosphorylation status of Src during the infection, were found to be influenced by the TPL arrangement. These results indicate the potential for novel host-pathogen interactions mediated by the TPL and SHB domains of AI NS1 protein. Δ225–230 protein, Δ221–230 protein, Avian influenza (AI) viruses pose significant threats to both animal and human health. Approximately 200 million birds have died or have been culled worldwide as a result of AI. In addition, since 1997, AI viruses of the H5 or H7 subtype have crossed the species barrier and caused fatal disease in humans PL domains are specific sequences, usually located in the last 5 to 6- C-ter aa of cellular proteins, that recognize the structural protein-protein interaction module PDZ Δ225–230, of the HPAI H7N1 viral strain, bearing only the PL domain II , resulted in the complete loss of interaction with the PDZ domains . To independently confirm that the interaction observed with the isolated PDZ domain of RIL on the arrays, reflected true protein-protein interactions, recombinant F4Δ225–230 and F6Δ221–230 proteins were used in pull-down assays with recombinant GST-RIL expressed in E.coli bound to GSH-sepharose beads. As shown in Δ225–230  of Src  for the F4Δ225–230, the ΔCF3Δ225–230 and the F6Δ221–230 proteins  of Src . The F4Δproteins 4 b. ColΔ225–230, or F6Δ221–230 proteins  with respect to the consensus, while all the H7N1 NS1 proteins retain a perfect match (-PPLPPK-). We cloned the NS1 protein from two seasonal H3N2 human influenza isolates of the 2007 epidemic (VR9220 and VR9240), bearing an intact SHB domain 1, but with one aminoacid substitution in a conserved Pro of SHB domain 2 (-PPØTPK-), an compared them to the F3 and H5N1 NS1 protein for their ability to stimulate Src. No stimulation of Src was observed by the full length H5N1 NS1, or by the two H3N2 NS1 proteins  proteins, respectively, with an A/turkey/Italy/977/99 H7N1 backbone  abolishes interaction with PTPL1 but not with RIL and Src. Deletion of the entire TPL (as in the F6Δ221–230 mutant) from the H7N1 NS1, abolishes the interaction with RIL, but still allows interaction of the viral protein with Src. Thus, the H7N1 NS1 protein seems to carry the potential for a wider range of interactions than its H5N1 counterpart, thanks to its TPL and SHB domain 2.Here, we showed that AI virus NS1 can interfere with the Src-RIL regulatory axis in a manner which is influenced by the PL arrangement. RIL was shown to bind to the phosphatase PTPL1, allowing its association with the active form of Src Δ221–230 mutant lacking all PL domains, was found to be sequestered into the insoluble fraction, upon lysis of infected cells, while the full length F3 NS1 protein was also retained in the soluble part, along with RIL and Src. The soluble fraction of active Src was indeed specifically enriched in infected cells expressing full length NS1, but not the F6Δ221–230 mutant. Src subcellular localization is subjected to complex regulation and Src has been found associated not only to the cytoplasmic side of the plasma membrane, but also to the endoplasmatic reticulum, Golgi apparatus and cytoskeletal components We propose a model, whereby AI NS1 replaces PTPL1 as the interacting partner of RIL, preventing Src inactivation. RIL binds to the SH2 and kinase domain of Src, allowing NS1 interaction with the SH3 domain. Binding of a protein substrate displaces RIL from the Src-NS1-RIL complex, whereby NS1 increases the catalytic efficiency of Src. Loss of the PL domain prevents NS1 interaction with RIL. Interestingly, the F6If loss or inactivation by mutation of the PL domain is a step involved in adaptation of AI viruses to new hosts, those strains bearing two PL domains in tandem (TPL), as those reported here, would present an higher genetic barrier to adaptation. The comparison of the PDZ-binding specificity of the F3 and F4 H7N1 NS1 proteins, indicated that the set of PDZ domains bound by the NS1 protein bearing both TPL I and II (F3), was significantly different from the one recognized by the NS1 protein bearing TPL II only (F4). This suggests that these two domains are not identical and that deletion of the TPL I can alter the network of interactions between AI NS1 and cellular PDZ proteins, possibly affecting pathogenesis. Thus, AI viruses bearing a TPL may represent a potential threat in case of cross-species infections A therapeutic strategy that can reduce the emergence of viral resistance towards antiviral drugs is the selective targeting of host factors required for viral replication. We have recently provided a successful example of such an approach towards HIV-1 infection 32P]- ATP was from GE Healthcare. All other chemical reagents were from Merck and Fluka.Labelled  (to detect the active form of Src kinase) or anti-Src [pY529] (to detect the inactive form of Src kinase) antibodies (Biosource). For detailed protocol see Supplementary Information.Alternatively, recombinant His-tagged F3 (20 µg) coupled to NiNTA-agarose beads in PBS 1× were mixed with HeLa cell lysate for 1 h at 4°C. Pulled down proteins were resolved on SDS-PAGE 10% gels. Samples were analyzed by Western blotting using either primary rabbit anti-Src  and in the presence of 0.012 µM of Src and 0.160 pmol of [γ-32P]ATP. Unlabelled ATP was added to reach the final concentrations as indicated in the figure legends. Abl activity was measured in a filter-binding assay using an Abl specific peptide substrate , 0.012 µM [γ-32P]ATP, 0.022 µM c-Abl. Reaction conditions were : 25 mM Tris-HCl pH 7.5, 1 mM DTT. Unlabelled ATP/Mg++ (1∶1 M/M) mix was added to reach the final ATP concentration of 100 µM. Reactions were incubated for 10 min at 30°C. Samples (9 µl) were spotted on paper cellulose filters which were washed according to the manufacturer's protocol. Filter-bound radioactivity was measured by liquid scintillation with a Microbeta-Trilux apparatus (Perkin-Elmer).Src activity was measured in a filter-binding assay using a commercial kit , according to the manufacturer's protocol, using the specific Src peptide substrate [5 human alveolar basal epithelial cancer cells  were infected following standard protocols for 48 hrs at an M.O.I. of 1 with either recombinant virus r977 or r4426 with the addition of 1.5 µg/ml of TPCK trypsin.For the western blot experiments, 1×10A549 cells were harvested following infection and incubated on ice for 30 min in 50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.35 M NaCl, 0.25% Triton in the presence of protease inhibitor cocktail. Cells were then centrifuged 10 min at 4°C (20000×g). The supernatant was kept as the soluble fraction. The pellet was extracted by sonication (3×5 s) in 0.5 M NaCl, supplemented with 1×Laemli Buffer, heated 5 min at 100°C, centrifuged (30 min 20000×g) and the supernatant kept as the insoluble fraction.The kinetic constants were derived by fitting the experimental data to the appropriate rate equations, as detailed in supplementary Information. Curves were obtained by non-linear least squares computer fitting of the data to the equations with the program GraphPad Prism 3.0.http://www.ebi.ac.uk/Tools/clustalw2/). Similarity searches were performed with the tools BLAST (http://www.expasy.ch/tools/blast/) and PROSITE (http://www.expasy.ch/tools/scanprosite/). Network interaction analysis was performed by STRING (http://string-db.org/).All sequence alignments were generated with the tool CLUSTALW Click here for additional data file.Figure S2Phylogenetic tree of the AI viral isolates bearing the novel TPL domain. The tree was generated starting from the aminoacidic sequences of 195/427 isolates present in the database, including all the sequences of the KRYMERRVESEI, KRYMARRIESEV and KRYMARRVESEI groups and 97/325 sequences of the most common KRYMARRVESEV group. Isolates with the same TPL domain version are grouped by coloured frames. The tree was generated by the platform Phylogeny.fr (http://www.phylogeny.fr/) with the program sMUSCLE, Gblocks and PhyML . Tree was drawn by the program TreeDyn.(PDF)Click here for additional data file.Figure S3Analysis of cellular PL type II domains. Sequence alignment of the human cellular PDZ-domains interacting with H7N1 and H5N1 NS1 proteins, identified by protein arrays analysis. Blocks of identical residues (in red) are highlighted in yellow. Similar aminoacids are highlighted in green. The derived consensus is shown (in red) at the bottom of the panel. Ø, hydrophobic residues; ¥, aliphatic residues. b. STRING interaction network of the cellular proteins bearing a PL domain type-II  and their respective PDZ-ligands . Colour coded connecting lines as per STRING nomenclature are as follows: purple, experimentally proven physical interactions; green, co-cited in the same article (PubMed); cyan, linked through functional homology. c Expanded STRING interaction network built from the cellular proteins (circled in red) with PL domain II  and their respective PDZ ligands . The thickness of the connecting lines is proportional to the STRING score (likelihood of interaction).(PDF)Click here for additional data file.Figure S4Phylogenetic tree of the NS1 proteins from H7N1 AI viruses isolated during the Italian 1999–2001 epidemic. The tree was generated using the neighbour-joining method as implemented by the MEGA4 program , Mol. Biol. Evol. 24, 1596–1599). Green circles identify isolates with a full length (230 aa) protein; yellow triangles identify isolates with a 6 aa C-ter truncation (Δ225–230); red squares identify isolates with a 10 aa C-ter truncation (Δ221–230).(PDF)Click here for additional data file.Table S1TPL an SHB2 domains of AI viruses grouped according to the corresponding hemagglutinin (H) subtypes.(PDF)Click here for additional data file.Table S2Cellular PDZ proteins interacting with H7N1 and H5N1 NS1 proteins.(PDF)Click here for additional data file.Table S3PDZ-binding human proteins bearing NS1 PL II homologous domains and their cellular ligands.(PDF)Click here for additional data file.Table S4Alignment of human PDZ domains interacting with cellular PL domain II-homologs, to the PDZ domain of the human protein RIL, interacting with AI NS1.(PDF)Click here for additional data file.


The extramembrane part of this enzyme consists of three domains: two domains related to the immunoglobulin fold and a catalytic domain belonging to the ErfK/YbiS/YhnG family at the C-terminus.The crystal structures of two fragments of the  Mt2 catalyzes the formation of the (3–3) cross-links characteristic of the peptidoglycan layer in the Mycobacterium tuberculosis cell wall. Bioinformatics analysis suggests that the extramembrane part of the enzyme consists of three domains: two smaller domains (denoted as A and B domains) and a transpeptidase domain (the C domain) at the C-terminus. The crystal structures of two fragments comprising the AB domains and the BC domains have been determined. The structure of the BC module, which was determined to 1.86 Å resolution using Se-SAD phasing, consists of the B domain with an immunoglobulin-related fold and the catalytic domain belonging to the ErfK/YbiS/YbnG fold family. The structure of the AB-domain fragment, which was solved by molecular replacement to 1.45 Å resolution, reveals that despite a lack of overall sequence identity the A domain is structurally very similar to the B domain. Combining the structures of the two fragments provides a view of the complete three-domain extramembrane part of LdtMt2 and shows that the protein extends at least 80–100 Å from the plasma membrane into the peptidoglycan layer and thus defines the maximal distance at which cross-links are formed by this enzyme. The LdtMt-related transpeptidases contain one or two immunoglobulin domains, which suggests that these might serve as extender units to position the catalytic domain at an appropriate distance from the membrane in the peptidoglycan layer.The transpeptidase Ltd These are the high level of amidation on the d-glutamate (d-Glu) and diaminopimelate (Dap) moieties and a high proportion (80%) of Dap–Dap (3–3) cross-links  cross-links are formed by d,d-transpeptidases, which are also called penicillin-binding proteins (PBPs). In contrast, the Dap–Dap (3–3) cross-links are created by l,d-transpeptidases  layer is responsible for rigidity and osmotic stability and provides the foundation of the cell envelope . LdtMt2 (Rv2518c) has been reported to be expressed at the highest level and has been found to be essential for virulence. Inactivation of this gene led to an attenuated phenotype and to increased susceptibility to clavulanate/amoxicillin treatment in vitro and in a mouse tuberculosis model  based on the X-ray crystallographic structures of two fragments of LdtMt2 representing the extramembrane part of the protein (residue range 55–408). The structure analysis reveals that LdtMt2 folds into three domains: two domains in the N-terminal part, both of which show an immunoglobulin-related fold, and a C-terminal transpeptidase domain. This domain composition is different from the two-domain structure of the extramembrane part of LdtMt2 proposed recently , providing an estimate of the maximal distance of the catalytic site from the membrane and thereby the approximate distance at which 3–3 cross-links are formed in the PG layer. Mass-spectrometric analysis provides further evidence that LdtMt2 forms covalent adducts with the β-lactam antibiotics imipenem and ampicillin.Here, we report the three-dimensional structure of Ldt2.2.1.Mt2  included amino acids 34–408, lacking only the predicted transmembrane domain (residues 18–34) and the intracellular part of the polypeptide chain (residues 1–17). Additional constructs were based on the domain arrangement obtained from a bioinformatics analysis of the protein sequence (see §a).The expression construct for the complete extramembrane segment of Ldtnce see §a.Pfu Turbo polymerase (Stratagene) with M. tuberculosis H37Rv genomic DNA as the template. Each construct was expressed in the pNIC28Bsa4 vector (GenBank accession No. EF198106) with a cleavable N-terminal His6 tag (H2N-MHHHHHHSSGVDLGTENLYFQ*SM). Proteolytic tag removal by TEV protease (the cleavage position is indicated by an asterisk) resulted in recombinant proteins with two additional amino acids (Ser-Met) at the N-terminus. All proteins were expressed in E. coli BL21 (DE3) cells at 294 K in LB medium supplemented with 30 µg ml−1 kanamycin. Gene expression was induced by the addition of 0.1 mM IPTG at an OD600 of 0.5–0.6 and the cells were harvested 20 h post-induction. The pellets were washed in 0.3 M NaCl, 10 mM Tris–HCl pH 8.0 and the cell walls were disrupted by flash-freezing and treatment with lysozyme (40 µg ml−1) and DNase I (2 µg ml−1) followed by sonication. The recombinant proteins were purified by affinity chromatography using His-Pur Ni–NTA resin (Thermo Scientific) and were eluted with an increasing imidazole gradient. Subsequently, the buffer was exchanged to 0.15 M NaCl, 25 mM Tris–HCl pH 8.0 using a desalting column . The His6 tag was removed by TEV protease cleavage with 5 µg TEV per milligram of recombinant protein at 293 K in the presence of 2 mM DTT. Cleaved proteins were separated from residual uncleaved proteins and TEV protease by a second Ni–NTA affinity step. Finally, a size-exclusion chromatography step was performed using a Superdex 200 column (Pharmacia Biotech) equilibrated with a buffer consisting of 0.15 M NaCl, 25 mM Tris–HCl pH 8.0. Pure protein samples were concentrated using a Vivaspin 20 centrifugal device  and the protein concentration was determined according to the Bradford assay using reagents from Thermo Scientific  and a BSA standard. The samples were analyzed by SDS–PAGE and native PAGE , aliquoted in the final buffer , flash-frozen in liquid nitrogen and stored at 193 K. The selenomethionine-substituted BC-module construct was produced via the metabolic inhibition method  and the commercially available crystallization screens PACT, JCSG+, NeXtal Classics II Suite (Qiagen) and SaltRX (Hampton Research). Crystals of the AB module were obtained in sitting drops at 293 K by mixing 0.15 µl protein solution and 0.15 µl well solution  and equilibrating against 50 µl well solution. The crystal used for data collection was flash-cooled in liquid nitrogen without using a cryoprotectant. The crystals of the BC module were grown in a hanging-drop format at 293 K by mixing 1 µl protein solution and 2 µl well solution  and equilibrating against 1.0 ml well solution. Crystals of the selenomethionine-substituted BC module were obtained in conditions identical to those for the native protein. The crystals of the BC module (native and selenomethionine-substituted) were cryoprotected by a short soak (20 s) in mother liquor supplemented with 30%(v/v) ethylene glycol prior to freezing. The crystals were frozen in nylon loops and stored in liquid nitrogen until data collection.Protein crystallization screening was carried out at protein concentrations of 17.5 mg ml2.3.XDS , Grenoble, France. In addition, a data set was collected to 2.7 Å resolution from a crystal of the SeMet-substituted BC module on beamline ID29 at the ESRF at the Se edge at a wavelength of 0.97677 Å. All data sets were collected at 100 K. The X-ray data were processed and scaled using C2, with unit-cell parameters a = 114.4, b = 27.6, c = 68.0 Å, β =114.1°, and contained one molecule in the asymmetric unit with a solvent content of 47.8%. Crystals of the BC module (native and selenomethionine-substituted) belonged to space group I212121, with unit-cell parameters a = 116.0, b = 121.4, c = 123.3 Å, and contained two molecules in the asymmetric unit with a solvent content of 68%. The statistics for all data sets are summarized in Table 1The crystals of the AB module belonged to space group 2.4.SHELX  and 30.1% (CCweak). An almost complete model was built by ARP/wARP . The coordinates of the AB module and BC module have been deposited in the Protein Data Bank under accession codes 4hu2 and 4huc, respectively.Both protein models were validated using 2.5.Mt2 (5 µg) were dispensed in water without antibiotics and with 2 mM imipenem (Sigma) or ampicillin  and incubated for 45 min at 295 K. Subsequently, the samples were diluted in 0.5 ml denaturing buffer  and loaded onto a CapLC system and an ESI-Q-TOF quadrupole/orthogonal acceleration time-of-flight mass spectrometer (Waters Corp.) using the method described by Sundqvist et al.  to obtain the molecular weights of the protein molecules and the protein–antibiotic adducts.Samples of Ldt al. 2007. The spe3.3.1.lppS gene (Rv2518c) encodes an enzyme consisting of 408 amino acids denoted LdtMt2. The polypeptide chain contains a short intracellular segment (residues 1–17), a predicted transmembrane domain (residues 18–34) and a larger extramembrane part (residues 35–408). The amino-acid sequence of LdtMt2 was analyzed with respect to potential domain organization using transmembrane-domain prediction  and several proteins derived from different corynebacteria genomes that also contain a predicted l,d-transpeptidase domain suggested that this fragment may be a separate domain. Furthermore, the sequence comprising residues 34–250 was subjected to domain-border analysis/prediction . Based on this analysis, three constructs were designed: a full-length construct (residues 34–408), the AB module (residue 55–146) and the BC module (residues 149–408).The E. coli and purified to homogeneity. Based on size-exclusion chromatography, the protein is a monomer in solution. However, extensive crystallization screens did not result in crystal formation. The AB- and BC-module constructs also resulted in soluble protein and each construct behaved as a monomer in solution.The full-length extramembrane fragment was expressed in 3.2.a). Six surface residues were modelled in double conformations . The model also contains 480 water molecules and 12 acetate ions, which mostly interact with surface residues. Two of the acetate ions are located in the active-site cleft of domain C. Finally, each polypeptide chain contains a bound metal ion. We modelled the bound metal as an Na+ ion based on the electron density (i.e. no remaining difference density after refinement), typical metal–ligand distances in the range 2.2–2.5 Å and the composition of the ligand sphere. The Na+ ion is located at the C-terminus of a surface helix and is coordinated by the carbonyl O atoms of residues 342 and 345 and by three water molecules.The crystal structure of the BC module was solved to 1.86 Å resolution by Se-SAD phasing from crystals of selenomethionine-substituted protein. The electron density for the two polypeptide chains (residues 149–408) in the asymmetric unit is well defined Fig. 2a. Six sb). The crystal asymmetric unit contains two molecules with a monomer–monomer interface of approximately 800 Å2, suggesting crystal-packing contacts rather than dimer formation. The structures of the two molecules in the asymmetric unit in the crystals of the BC module are essentially identical, displaying a Cα r.m.s.d. of 0.3 Å for 259 aligned residues. The overall structure of this module and the interactions of the two polypeptide chains in the asymmetric unit are very similar to a recently determined structure of a corresponding module of LdtMt2, albeit obtained from a different expression construct  that protrude from the core of the catalytic domain  and the residues surrounding the entrance to the active site  contribute to the closure and to the exclusion of solvent, which may support efficient catalysis.The core of the transpeptidase domain consists of a β-sandwich formed by two β-sheets packed together. Comparison of this domain with a related protein from in Fig. 3. This feb). The three tryptophan residues  in this segment form extensive stacking interactions at the domain interface and are likely to stabilize the relative orientation of the two domains. The pattern of tryptophan residues in this sequence stretch is retained in Rv0483 (LprQ), one of the homologues from the M. tuberculosis H37Rv genome.Residues 382–408 form a C-terminal extension which is not present in the two characterized structural homologues. This sequence stretch runs along the C domain and contributes to the domain interface by forming additional contacts with the B domain Fig. 1b. The tet al., 2012b) related to the immunoglobulin-like fold .As has been noted previously . Seven residues, Val59, Val66, Asn121, Arg123, Thr138, Glu162 and His214, were modelled in double conformations. Each domain in the model contains a bound sulfate ion located on the protein surface. In domain A the sulfate is anchored to the protein through interactions with Asn95, Asn97, Arg99 and Arg122, whereas in the B domain the ligand forms hydrogen bonds to the side chains of Tyr201, Arg209 and Arg211.The structure of the segment comprising the A and B domains was solved to 1.45 Å resolution by molecular replacement using the B domain (residues 149–250) as the search model. The structure of the AB module includes residues 57–250, approximately half of the extramembrane part of Ldted Fig. 2b. Sevenc and 4et al., 1994Mt2 belong to the c-type Ig fold, with strand orders a–b–e–d and c–f–g in the two antiparallel β-sheets  found between β-strands β2 and β3, which is not present in domain A, and the loop connecting β6 and β7, which is substantially longer in the B domain . The sequence identity derived from structural alignment of the two domains is 12% which does not provide strong evidence for gene duplication.The similarity between the A and B domains is reflected in the Cn Figs. 4a. The s2 of buried surface area including 13 and ten buried residues from the A and B domains, respectively. The domain interface is formed by the loops connecting β-strands β1 and β2 and β-strands β5 and β6 in domain A and the loop connecting β-strands β6 and β7 in domain B. The two domains are connected by a short linker Ala149-His150 forming hydrogen bonds to the main chain of the B-domain residues involved in the interface (Gly236 and Phe237). The size of the AB-domain interface, the short linker and the fact that B factors of the linker residues are in the range 18–22 Å2 as in the remaining part of the structure suggest that the domain linker is not flexible and the orientation of the two domains is kept in this V-shaped form . The structures of the B domain in the AB and BC modules are essentially identical and superposition gives an r.m.s.d. of 0.4 Å for 101 aligned residues, although the B-factor profiles for these domains are different in the two crystal forms, most likely owing to different crystal-packing interactions.The domain interface accounts for 350 Årm Fig. 1c. The sMt2 and the results presented here redefine the domain composition of this enzyme. A recent structure analysis described the extramembrane part of the protein as a two-domain structure extending from the membrane  which had not been recognized previously. The extramembrane part of the polypeptide chain of LdtMt2 is thus a three-domain entity, with two immunoglobulin-like domains rather than one and a C-terminal transpeptidase domain.The two constructs used in the crystal structure analysis cover approximately the whole length of the extramembrane part of the polypeptide chain of Ldtus Fig. 1c which DALI identified the closest structural homologues of the two immunoglobulin-like domains as the periplasmic copper-resistance protein from Pseudomonas syringae  and the N-terminal domain of Halothermothrix orenii amylase  and Cα r.m.s.d. values (2.6–3.1 Å). Finally, the cell-wall-located S-layer protein from Geobacillus stearothermophilus  and with the homologous domains of the mannanase (18.5%) and a glucodextranase from Arthrobacter globiformis (15.2%)  could potentially be relevant as the PG backbone is a duplex of carbohydrate chains. In the multi-domain amylase enzyme from H. orenii it has been experimentally demonstrated that the small noncatalytic immunoglobulin-like domain mediates the interaction of the enzyme with the high-molecular-weight substrate .A structural comparison using 3.4.Mt2 and contain an overlapping domain (domain B), the structure of the full-length protein excluding the short intracellular and membrane-stretch parts can be reconstructed by superposition of the AB and the BC modules based on the B domains  to the BC module of LdtMt2. On the other hand, Rv0483 and Rv0192 appear to have a three-domain arrangement as in LdtMt2 as they not only show sequence homology to the B and C domains but also to the A domain (35% sequence identity for Rv0483 and 18% for Rv0192). The five l,d-transpeptidases in the M. tuberculosis genome thus can be divided into two groups with one or two immunoglobulin-like domains between the plasma membrane and the catalytic domain. The variation in the number of these domains positions the catalytic domain at different distances from the membrane, reflecting the multilayered structure of the peptidoglycan in the bacterial cell wall. These domains define the distance of the catalytic site and thus the site of formation of the 3–3 linkages in the PG relative to the membrane. Cross-link formation appears to occur at two levels carried out by the BC module-like l,d-transpeptidases (Rv0116c and Rv1433) and by the one domain longer LdtMt2-type , a homologue of LdtMt2  and ampicillin  led to the formation of covalent enzyme–antibiotic adducts as shown by ESI-MS (Table 2M ampicillin or 2 mM imipenem corresponds to the exact mass of the respective antibiotic (Table 2Mt2 using ITC (Erdemli et al., 2012Mt2.The inhibitory effect of β-lactam-type antibiotics towards transpeptidases is based on the formation of a covalent adduct with the active-site serine residues in c Table 2. TogetheMt2, AB module, 4hu2LtdPDB reference: BC module, 4hucPDB reference: 10.1107/S0907444912049268/wd5198sup1.pdfSupporting information file. DOI:


A new method for the classification of domain movements in proteins is described and applied to 1822 pairs of structures from the Protein Data Bank that represent a domain movement in two-domain proteins. The method is based on changes in contacts between residues from the two domains in moving from one conformation to the other. We argue that there are five types of elemental contact changes and that these relate to five model domain movements called: “free”, “open-closed”, “anchored”, “sliding-twist”, and “see-saw.” A directed graph is introduced called the “Dynamic Contact Graph” which represents the contact changes in a domain movement. In many cases a graph, or part of a graph, provides a clear visual metaphor for the movement it represents and is a motif that can be easily recognised. The Dynamic Contact Graphs are often comprised of disconnected subgraphs indicating independent regions which may play different roles in the domain movement. The Dynamic Contact Graph for each domain movement is decomposed into elemental Dynamic Contact Graphs, those that represent elemental contact changes, allowing us to count the number of instances of each type of elemental contact change in the domain movement. This naturally leads to sixteen classes into which the 1822 domain movements are classified. From a structural perspective domains in proteins can be regarded as quasi-globular regions. The connections between domains allow their relative movement and consequently domain movements are often engaged in protein function In an influential review of protein domain movements using structures from the PDB, Gerstein et al. The method presented here is based on changes in interdomain residue contacts that occur in the domain movement. The advantage of such a method is that it is relatively simple to implement but has a connection to methods based on calculating interaction energies. Key to the analysis is the concept of the “Dynamic Contact Graph” (DCG). Each domain movement has an associated DCG. Using graphs has three benefits: they provide a visual metaphor for the movement they represent; they provide motifs for some basic domain movements that are instantly recognisable; the well-developed algorithms of graph theory can be used to evaluate features of interest. The analysis is developed in terms of “elemental” DCGs which represent elemental contact-changes. These elemental contact-changes can, under certain assumptions, be associated with “model” domain movements. We count the number of elemental DCGs any general DCG comprises which naturally leads to sixteen different categories into which the domain movements are classified. The results are presented at a website.http://www.cmp.uea.ac.uk/dyndom. In order to simplify the analysis only those cases with two domains were used. Of the 2035 cases, 1822 are two-domain proteins. The two domains in each protein will be referred to as “domain A” and “domain B” below.The basic data are the 2035 unique domain movements from the NRDPDM “Contact” between residue i and residue j means any heavy atom of residue i is within 4 Å of any heavy atom of residue j. However, before the set of pair-wise contacts between residues in each domain and for each conformation is determined, residues at the boundaries of the domains annotated by DynDom as bending regions were removed as were residues close to the interdomain screw axis (any heavy atom of the residue within 5.5 Å of the axis). The reason for this is that they would be expected to have maintained contacts (see below) irrespective of the nature of the domain movement.1,b1) denote a “residue contact pair”, where a1 is the residue number of a residue in domain A, and b1 is the residue number of a residue in domain B, that make contact in conformation 1. Similarly let  represent a residue contact pair in conformation 2. By considering at most a single residue contact pair between the domains in either conformation there are five “elemental contact-change” scenarios (where below  indicates no contact exists):Let   =  and   = .“new”: either  ≠ and   =  or   =  and  ≠.“maintained”:  ≠ and  ≠ where a1 = a2 and b1 = b2.“exchanged-partner”:  ≠ and  ≠ where (a1 = a2 and b1≠b2) or (a1≠a2 and b1 = b2).“exchanged-pair”:  ≠ and  ≠ where a1≠a2 and b1≠b2.“The contact-changes can be associated with five “model” domain movements assuming the following idealisation.The domains have a spherical shape and are perfectly rigid.There is only one residue from each domain at a contact point.The relative movement of the domains is a rotation about a hinge axis passing through an interdomain linker region which is short in comparison to the size of the domains.free”. The “new” case implies the domains move from a contacting to non-contacting conformation (or vice-versa) suggesting a rotation about a hinge axis perpendicular to the line joining the centres of mass of the domains, defined previously as a “closure” motionopen-closed” movement. The “maintained” case means the domains cannot move (given that we exclude the hinge region which would otherwise be designated as maintained region) implying the domains remain “anchored”. For the “exchanged-partner” case we have the same residue from one domain making a contact in both conformations but with different residues on the other domain. This implies one domain sliding over the other and is easiest to imagine occurring by a relative twist of the domains. Consider the hinge axis passing through the centre of mass of domain A, with the centre of mass of domain B slightly shifted from the hinge axis, i.e. predominantly a twist motion sliding-twist”. For the “exchanged-pair” case, the two residues making contact in one conformation are not involved in making contact in the other conformation again implying a movement with the hinge axis perpendicular to the line joining the centres of mass. The movement would break the contact on one side of the domains and rotation continues until contact is made on the other side of the domains. This is commonly known as a “see-saw” motion which has already been seen to occur in lactoferrin The “no contact” case implies the domains remain separated and can move freely. This case we call “The association of these elemental contact-changes with the model domain movements is based on consideration of the simplest, most plausible domain movement to reproduce the elemental contact-change in an idealised system. In reality even in those cases where only one type of elemental contact-change occurs, the movement might not resemble the corresponding model domain movement as domains are not perfectly rigid and often have complex interfaces. The extent to which real domain movements conform to these idealised movements is something to be determined.1i,b1i)}, i = 1,N1 denote the set of residue contact pairs in conformation 1 and {}, i = 1,N2 the corresponding set for conformation 2.Here we introduce Dynamic Contact Graphs (DCGs). Let {(a1i→b1i. For contacts in conformation 2 the direction is from the residue (node) in domain B to the residue (node) in domain A . This could be written as a2i←b2i. Each node of the graph represents a residue of which there are two types: those in domain A and those in domain B. An edge exists when there is a contact between a residue in domain A and a residue in domain B, i.e. when they appear in one of the sets above. The key feature of a DCG is that it is directed. For contacts in conformation 1 the direction associated with an edge is from the residue (node) in domain A to the residue (node) in domain B . This could be written as aIn general a domain movement may combine these elemental contact-changes and have a complex graph structure.We make full use of Matlab (version 8.0.0.783 (R2012b)) and in particular the Bioinformatics Toolbox “biograph” function to create a “biograph” object, a data structure for directed graphs. This enabled us to use associated methods to analyse and view the DCGs.Information on each domain movement can be found at our website. Each domain movement has its own webpage on which its DCG is shown. However, 413 domain movements have no contacts in both conformations (apart from at the removed hinge regions). For these the DCG is empty. These domain movements are assigned to the “no contact” class which implies a free movement of the domains.The remaining 1409 domain movements each have a DCG. An illustrative example from a DNA topoisomerase III is shown in As can be seen in maint represent the number of maintained-changes. For the DNA topoisomerase III shown in maint = 7. Counting exchanged-partner contact-changes is not unambiguous as illustrated in Our aim is to count the number of contact-changes of each type for each domain movement. This is equivalent to decomposing a DCG into the four elemental DCGs shown in The problem of identifying exchanged-partner contact-changes is therefore equivalent to finding the maximum number of non-overlapping triplets in the DCG.Once the maintained and exchanged-partner contact-changes have been assigned the exchanged-pair and new contact-changes are assigned as detailed below.The first step counts the number of maintained contact-changes in a DCG and then creates a new DCG that has no double links. The maximum number of non-overlapping triplets in the resulting graph was then determined as follows. First all possible triplets (overlapping and non-overlapping) were determined. A new (undirected) graph was then created which had a node (vertex) for each triplet and an edge between any two nodes with triplets that overlap. An exhaustive search was implemented to find the maximum number of non-overlapping triplets. The algorithm involved selecting a node, removing those nodes connected with it by a single edge and repeating this process until no nodes remain. The selected nodes give a set of non-overlapping triplets. This recursive program is given here in pseudo-code:Input: A graph with vertices  ordered,V = v1,v2,v3,.. ,vnand a set of edgesE(an edge existing if the two vertices represent triplets that overlap).Output: A list of vertices,Wmax, with the maximum number of vertices,Nmax, none of which are connected by a single edge.maxN = 0maxW = {}W = {}addv1toWw = v1V’ = Vunconnected{if (|V’| = 0){if (|W|>maxN){maxW = WmaxN = |W|}returnmaxW,maxN# terminate branch in search tree if it cannot# exceed maxN}elseif (|V’|+|W|< = maxN){return}w,jv)ϵE) {while }w, add to W; remove vertices with an edge connecting to w; continue first two steps until exhaustion of vertices. Then search amongst the N W recorded for each repetition for maxN and maxW). This random search found the same value of maxN determined by the exhaustive search in all 1397 DCGs that could be search exhaustively. Nexchpart, the number of exchanged-partner contact-changes is set equal to maxN.For twelve DCGs this exhaustive search was too slow and was replaced by a related random search . In a DCG with maintained, exchanged-partner and exchanged-pair contact-changes removed there are only two-node subgraphs of one type left, either AB or BA. These represent the new contact-changes. The number of new contact-changes, Nnew, is then given by Nnew  = n1- Nexchpair or Nnew  = n2- Nexchpair, the former if n1≥n2, the latter if n2>n1.A DCG with maintained and exchanged-partner contact-changes removed comprises disconnected two-node subgraphs. Each subgraph has a single AB edge for conformation 1 or a single BA edge for conformation 2 and these are paired off to count the number of exchanged-pair contact-changes. Let nmaint = 0, Nexchpart  = 2, Nexchpair  = 0 and Nnew = 0. For the less trivial case of DNA topoisomerase III shown in maint = 7, Nexchpart  = 13, Nexchpair  = 7 and Nnew = 4.For the example in We classify domain movements according to which of the contact-change categories are non-empty or empty. There are five types of contact-change, but given that for all domain movements there are always residues that do not make interdomain contacts in both conformations, the no contact-change case is redundant. The only interesting case is when all residues are in this category but this case is covered when the number of contact-changes in all the other categories is zero. Therefore we need only consider the remaining four contact-change categories.4) different classes. The no-contact class is when all four categories are empty. There are four “pure” classes, when only one category is non-empty, the other three being empty, e.g. “pure new” has Nmaint = 0, Nexchpart  = 0, Nexchpair  = 0, Nnew≥1. There are six classes when two categories are non-empty and two empty, e.g. “combined maintained, new” has Nmaint≥1, Nexchpart  = 0, Nexchpair  = 0, Nnew≥1. There are four classes when three categories are non-empty and one empty, e.g. “combined exchanged-pair, exchanged-partner, new” has Nmaint = 0, Nexchpart ≥1, Nexchpair ≥1, Nnew≥1. Finally, there is one class when all four categories are non-empty. These classes are given in Each of the four categories can be empty or non-empty meaning there are sixteen (2It is interesting that there are so many examples of domain movements where no contacts are made between the domains (except at the hinge bending sites) in both conformations. Some of these may be due to domain linkers that act as rigid spacers between the domains to prevent unfavourable interdomain interactions during folding In terms of the total number of contact-change types across the whole set, there are 6810 new, 6087 maintained, 1448 exchanged-pair and 1150 exchanged-partner contact-changes.http://www.cmp.uea.ac.uk/dyndom/class16). Each class comprises a list of protein names together with a pair of PDB accession codes and chain identifiers that specify the domain movement. The link provided takes one to a page where the DCG and a bar chart for the distribution of the number of instances in each of the four elemental contact-change categories are shown (see http://jmol.sourceforge.net/), is used to display the movement and to indicate the residues that make contact in each conformation. There is also a link to the corresponding DynDom page for that domain movement which gives details on the residues comprising the domains, the location of the hinge axis, the hinge-bending residues, the angle of rotation, percentage closure, as well as many other details, and a downloadable script for viewing the movement. A link to the DynDom family page is also provided which gives a conformational analysis of closely related structures and their domain movements We have produced a website where the domain movements are organised according to class , shows that an exchanged-partner contact-change can occur without a sliding twist movement if the interdomain screw axis is remote from the interdomain region, i.e. it violates one of the conditions for a model domain movement. A sliding twist movement is seen, however, in an immunoglobulin protein in the combined exchanged-partner, new class (PDB codes: 1E4K and 2IWG) where the domain movement is predominantly a twist (37% closure).Finally in the pure exchanged-pair class which is associated with the see-saw model domain movement, six out of the nine examples would conform to a see-saw movement in that one can find a plane that the interdomain screw axis lies in and for which the contacts in the two conformations occur on either side of this plane. An example can be see for a histidine kinase (PDB codes: 1B3Q and 2CH4) which undergoes a clear see-saw movement with the domains rotating through 126°. An example that would not seem to be like a see-saw movement can be see for a lytic transglycosylase (PDB codes: 2G6G and 2G5D) where the non-globular shape of the domains and their location in relation to the hinge axis allows an exchanged-pair contact-change to occur via a non see-saw-like movement.We have used a contact analysis to help classify domain movements in proteins. The approach introduced here is based on identifying five types of elemental contact-changes. A real domain movement will comprise these elemental contact-changes but decomposing contact-changes in a real domain movement into the elemental contact-changes is non-trivial. A solution to this problem was found by encoding the contact-changes in a DCG and decomposing it in terms of the elemental DCGs which represent the elemental contact-changes. This allowed us to count the number of instances of each of the elemental contact-change types for each domain movement. This in turn has led to a classification system comprising sixteen classes.Each elemental contact-change type can be related to a model domain movement. However, although some of those classified as “pure” in By counting disconnected subgraphs in a DCG, we are able to give the number of independent regions, that is, regions comprising sets of residues between which there are no contacts in either conformation. These regions may have a different role to play in the mechanism of the domain movement.The elemental contact-change types may relate qualitatively to the energetics of domain movements. The no contact class suggests no energy need be expended in the movement (except perhaps in the hinge bending region). The “new” type suggests energy needs to be inserted into the system or is expended. A “maintained” type suggests a strong interaction with little or no energy consumed or expended or perhaps energy being consumed or expended to strain or relieve a maintained bond. An “exchanged-partner” type may suggest a low energy barrier if a sliding movement occurs because as one interaction is weakened the other is being strengthened. An “exchanged-pair” type by contrast may indicate an energy barrier if one interaction is broken before the other one is formed in a see-saw movement. However, many domain movements are highly complex and this kind of simple interpretation will obviously not always apply. Indeed, one can imagine the exchanged-pair contact-change occurring in a way much like the sliding case if as one pair of contacts is being lost another pair of contacts is being gained such that there is no appreciable energy barrier. The work by Sinha et al. For enzymes it has been shown that the type of structural change can relate to the type of reaction being catalysed Although we have used the DCGs to classify domain movements, they should provide, in themselves, a great deal of insight in individual cases, especially when considered by experts on the protein concerned. In essence they give a visual metaphor for the movement and its mechanism. Here we consider motifs that appear in DCGs indicating particular mechanisms.Multiple new: A residue with no contact in one conformation moves into a pocket making multiple contacts in the other conformation. The associated graph is shown in Linear Interlocking: A sequence of interlocking residues as depicted in Anchoring residue: A single residue maintains contact with a number of other residues during the domain movement, acting perhaps as an anchor as shown in Linear slide: A region from domain B  simulation and Normal Mode Analysis (NMA). In the case of NMA a single normal mode eigenvector can be represented by two structures from which residue contacts or perhaps energy-based thresholds could be used to define the DCG. Likewise in the case of MD simulation principal component analysis gives eigenvectors from which two extreme structures can be created.DCGs provide us with a way to identify motifs related to movements of domains. However, DCGs need not be confined to the analysis of domain movements but can be applied to any case where there are two conformations and two sets of objects e.g. subunits that have different associations in the two conformations.


The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines . Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at  In recent years the Non-Ribosomal Peptide Synthetases (NRPSs) and PolyKetide Synthases (PKSs) have gained considerable interest as they can produce polypeptide- and polyketide-based secondary metabolites that exhibit important pharmaceutical and biological activities see e.g. . The SynEscherichia coli fatty acid synthase (FabD) and its substrate, enabled the identification of 13 active site residues in the AT domain The structure and activity of the natural products produced by NRPSs and PKSs are determined by the specific substrates that are bound by the A and AT domains, respectively. Co-crystallization of the malonyl-CoA-specific acyl-transferase [PDB∶1MLA] from Most substrate specificity prediction tools that have been developed are based on the A and AT active site residues. The tools include: NRPS–PKS The classification and selection procedures described above rely on multiple sequence alignment followed by clustering/classification through Neighbor-Joining (NJ). Initial attempts to cluster the A-domains according to substrate specificity using their whole sequence and the NJ-algorithm were only partly successful Although the tools perform well in predicting the substrate specificity of the AT and A domains for many substrates, for some substrates they perform less well http://www.ncbi.nlm.nih.gov] and UniProt http://www.uniprot.org]. The list of sequences and the appropriate literature references are given in sheet 1 of http://www.nrpssp.com] . For reasons of comparison, the previously identified sets of characteristic residues according to http://weblogo.berkeley.edu/].The residues of the aligned AT and A domain sequences were numbered according to the AT domain of It appeared that both the AT and A domain data-sets contained many duplicate or near duplicate sequences. To ensure a balanced coverage of the available sequence space, we removed the (near) duplicate sequences. In this way a non-redundant set of 167 AT domain sequences and 571 A domain sequences remained, as indicated in sheet 2 of http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/]. The tool provides the opportunity to paste or upload domain sequences and select the appropriate HMMs to analyze these sequences. To ensure a proper prediction it is essential to use only the sequence of the A or AT domain from the complete NRPS or PKS sequence, respectively. To identify and extract the domain boundaries from the protein sequence of the complete system we advise to use the search domain option in either of these tools NRPS–PKS http://www.cmbi.ru.nl/bamics/supplementary/Khayattetal_2012_NRPSPKS/. A representation of the presented workflow can be found in The HMMs were implemented in a straightforward manner using Python. The associated web-tool can be found at  are given. In sheet 2 the results of the HMM analysis are given. The annotation data related to supposedly wrong predictions and predictions below threshold were looked up and evaluated. Sheet 3 summarizes the analysis results. Sheet 4 provides an overview of the sequences that were present in both the non redundant data-set as well as the data-set from (XLSX)Click here for additional data file.


Euoplocephalus tutus (to investigate effects on our understanding of intraspecific variation) and Minotaurasaurus ramachandrani . The ratio of orbit maximum rostrocaudal length to perpendicular height is used as a strain ellipse, which can be used to determine if ankylosaur skull fossils have been dorsoventrally compacted during fossilization and diagenesis. The software program Geomagic is used to retrodeform three-dimensional (3D) digital models of the ankylosaur skulls. The effects of sediment compaction are modeled using finite element analysis, and the resulting strain distributions are compared with the retrodeformed models as a test of the retrodeformation method. Taphonomic deformation can account for a large amount of intraspecific variation in Euoplocephalus, but finite element analysis and retrodeformation of Minotaurasaurus shows that many of its diagnostic features are unlikely to result from deformation.Taphonomic deformation can make the interpretation of vertebrate fossil morphology difficult. The effects of taphonomic deformation are investigated in two ankylosaurid dinosaur taxa,  Variation among specimens referred to a single fossil taxon can originate from several biological sources, such as ontogeny, sexual dimorphism, and individual variation, but taphonomy can also be a source of morphological variation in fossils. During fossilization and diagenesis, bones can become deformed, and this deformation can lead to difficulties in understanding taxonomic variation, phylogenetic relationships, and functional morphology Fossils can become distorted from the effects of brittle or plastic deformation (or both). In geological terms, brittle deformation results in fractures, joints, and faults, and plastic deformation results in folds. Whether or not a fossil undergoes brittle or plastic deformation is dependent on the temperature, confining pressure, and strain rate it experiences. Brittle deformation occurs at low temperatures, low confining pressures, and high strain rates; plastic deformation occurs at high temperatures, high confining pressures, and low strain rates. Many fossils undergo brittle deformation prior to burial, cracking and fracturing during transport, and brittle deformation can occur during diagenesis as well, such as if a fossil is faulted. Plastic deformation of a fossil is more likely to occur during fossilization and diagenesis, during which time bone can act like a ductile material. Fossils rarely survive more than a single phase of plastic deformation, and as such, identifiable but plastically distorted fossils typically have a simple deformation history The goal of this study is to introduce some techniques for understanding three-dimensional (3D) plastic deformation in ankylosaurid dinosaur skulls. First, skulls of extant vertebrates were examined to determine if the shape of the orbit can be used as an indicator for whether or not plastic deformation has occurred. If the periorbital rims of a variety of extant vertebrates are generally circular, then fossil skulls with elliptical orbits have probably undergone some amount of plastic deformation. Retrodeformation and finite element analysis were then used as tools for understanding what parts of an ankylosaur skull are most likely to undergo deformation and therefore least likely to be phylogenetically useful. This information can then be used to enhance the quality of cranial characters used in phylogenetic analyses. No attempt was made to undistort taphonomically distorted skulls into their original shape, as there are few features on the skull to act as constraints guiding the decisions in retrodeforming ankylosaur skulls. Retrodeforming an ankylosaur skull with the goal of restoring its true shape would be highly subjective. Instead, the focus of this study is on understanding which morphological features on an ankylosaur skull are most likely to become taphonomically deformed.The software program Geomagic Euoplocephalus tutus Lambe, 1910 Minotaurasaurus ramachandrani Miles and Miles, 2009 Euoplocephalus  will highlight the changes that can occur during crushing, and can be used to identify areas of the skull that are most likely to change and therefore be less taxonomically informative. For example, if the erectness of the squamosal horns changes with retrodeformation, or if there is high strain in this area after FEA, then the erectness of the squamosal horn may be affected by dorsoventral compaction.This study examines two cases where understanding deformation can be used to better interpret ankylosaur cranial morphology: 1) intraspecific variation in cephalus  is the bMinotaurasaurus  was CT scanned at the University of Alberta Hospital ABACUS Facility. CT scans of the holotype of Minotaurasaurus (INBR 21004) were provided by V.S. Ramachandran . L. Witmer (Ohio University Heritage College of Osteopathic Medicine) provided CT scans of AMNH 5405, , which were originally published in Witmer and Ridgely Three ankylosaur skulls were converted into 3D digital models from computed tomography (CT) scans. UALVP 31  were imported into the software program Geomagic and retrodeformed using the Deform Region tool  are unknown, and the provenance of the holotype of Minotaurasaurus is unknown. Finally, each of the models were put through five different analyses  files and imported into the software program Strand7 analyses  approximOrbit shape measurements of extant taxa , Fig. 3 Euoplocephalus skull is bilaterally asymmetrical, but the arched profile in lateral view suggests that the skull has not been dorsoventrally compacted. Surprisingly, the orbit ratios  are higher than what would be expected if the skull was not crushed at all  is low and flat in lateral view and is nearly symmetrical. The orbit ratios are 1.72 (right) and 1.43 (left), which is slightly higher than what would be expected based on the survey of extant skulls. The orbits are also teardrop-shaped, which suggests that the skull may have been dorsoventrally compressed. Retrodeforming the skull in Geomagic resulted in an arched rostrum similar to that of AMNH 5405, more horizontally projecting squamosal horns, and more ventrally projecting quadratojugal horns , and the range of orbit ratios was much greater for ankylosaurs than for all extant taxa combined. A plot of ankylosaur orbit ratios  have noticeably different orbit ratios for the left and right orbits, which suggests that the skulls underwent shearing or uneven dorsoventral compaction. Orbit ratios may be most useful when compared across multiple specimens of the same taxon, and very high ratios above 2  are likely to indicate that dorsoventral crushing has occurred. The orbit ratio can serve as a general indicator if an ankylosaurid skull has been dorsoventrally compacted, but cannot be used to definitely indicate how much compaction has occurred. The true orbit ratio may not be known for a given fossil taxon, but high orbit ratios relative to the mean for a given sample of fossil specimens could also be used to identify if dorsoventral compaction has occurred. The orbit ratio could be a useful indicator of compaction for skulls that are symmetrical and which may not be obviously deformed.The higher orbit ratios in the few crocodilian and avian taxa in this study (representing the extant phylogenetic bracket for ankylosaurs) may suggest that archosaurian orbits are less circular than those of mammals, and that undeformed orbit ratios from 1.3–1.7 could be expected for dinosaurs. However, many of the ankylosaurid skulls had orbit ratios well above the maximum undeformed ratio recorded in this study . The FEA tests , 6 showeEuoplocephalus. For example, Penkalski Euoplocephalus have a range of morphologies in lateral view, from arched , to flat . It is possible that the arching of the skull may be related to ontogeny, in which case a correlation between flatness and size would be expected. It is also possible that the relative flatness may be a true taxonomic difference. However, many of the skulls that are flat also have subcircular orbits, which suggests that the skulls have undergone crushing and in life were more arched.Taphonomic distortion may be responsible for some of the variation in skulls referred to Minotaurasaurus as being flatter or more horizontal than their equivalents in other ankylosaurids: the angle of projection of the jugal horns, the articular surface of the quadrate, the pterygoid-quadrate contact, and the orientation of the pterygoid body. Additionally, the ‘flaring’ narial osteoderms may be a product of dorsoventral crushing. Retrodeformation of INBR21004 in Geomagic resulted in more ventrally projecting quadratojugal horns, but did not affect the quadrates or pterygoids (Minotaurasaurus are a result of deformation or represent true taxonomic differences. The flaring appearance of the narial osteoderms did not change during retrodeformation (Minotaurasaurus are real, and not an artifact of preservation.Miles and Miles erygoids . Howevererygoids . This suormation , and dorormation . In the ormation . This suAlthough Geomagic contains tools that could be used to correct plastic deformation in a fossil, there are many challenges associated with reconstructing a distorted fossil into its true, original shape. It is difficult to determine the accuracy of the retrodeformed skull in which there is no extant, undeformed analog. Simply restoring symmetry is insufficient to determine if a retrodeformed skull represents an accurate shape. Boyd and Motani Minotaurasaurus did not change during retrodeformation, which suggests that these features are either unique to this genus or represent intraspecific or ontogenetic variation within a different taxon. Much of the variation in skull morphology in specimens referred to Euoplocephalus may also be a result of taphonomic distortion, although again intraspecific and ontogenetic variation cannot be ruled out.Retrodeformation of a specimen may result in new taxonomic interpretations because of changes in shape. The accuracy of 3D retrodeformation techniques is still being investigated; retrodeformation is more likely to be successful when morphological constraints, based on features of extant taxa, can be used


Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog  Krüppel-type C2H2 zinc finger (ZNF) proteins with a N-terminal Krüppel-associated box (KRAB) form the largest family of potential transcription factors encoded in the human genome with about 400 members The KRAB domain is a protein-protein interaction domain. It is sufficient to confer transcriptional repression in heterologous reporter assays when tethered to a DNA binding domain The KRAB domain can be subdivided into 2 subdomains that are usually encoded by separate exons. The KRAB-A domain is a prerequisite for efficient transcripitional repression in heterologous reporter assays and for interaction to TRIM28 Xenopus laevis protein containing a remarkable large number of 37 C2H2 zinc finger motifs XFIN, a In this study, we compared the properties of the XFIN and PRDM9 KRAB domains with the KRAB domain of human ZNF10/Kox1 as “gold standard”. The results showed that the corrected XFIN KRAB-AB domain displayed considerably weaker transcriptional repressor activity than ZNF10 KRAB-AB. The main reason was the weak boostering ability of XFIN KRAB-B as shown by domain swapping experiments. These differences in repression activity coincided with the different extent of interaction with TRIM28. In contrast to ZNF10 and XFIN, neither the KRAB domain-related domain of PRDM9 nor its whole N-terminal part conferred transcriptional repression in reporter assays. Our study contributes functional data on previously poorly characterized KRAB domains of ancient evolutionary origin.Table S1 lists the sequences of the oligonucleotides used in the following recombinant DNA constructions. Fragments resulting from PCR were sequenced for verification. The eukaryotic expression vector pN2-GST was constructed as follows: The coding sequences for EGFP were removed from pEGFP-N2 (Clontech) by BamHI/NotI digestion and the backbone religated with a double-stranded oligonucleotide forming appropriate BamHI/NotI overhangs. The coding sequence for glutathione S-transferase from Schistosoma japonicum (GST) was amplified by PCR from pGEX-6P-1  and cloned as a HindIII/SalI fragment into this modified pEGFP-N2 to get pN2-GST. KRAB domain encoding sequences were inserted downstream of the GST cassette using XhoI/SalI. The respective DNA fragments were generated by PCR using templates for human ZNF10/Kox1 . XFIN KRAB-A was cloned by PCR from the AB-part. Fragments encoding seamless domain swaps between ZNF10-AB and XFIN-AB domains (ZNF10-A-XFIN-B and XFIN-A-ZNF10-B) were obtained synthetically through a commercial service . In parallel, the KRAB-encoding sequences were inserted as XhoI/SalI fragments into the pM3 effector plasmid. pM3 is an eukaryotic expression vector encoding N-terminally the DNA binding domain of the yeast transcription factor Gal4  was cloned by RT-PCR from human testis RNA (Clontech) and different fragments were generated by PCR and also inserted by XhoI/SalI into pM3. The expression vector for human TRIM28, pCMV-TIF1beta-flag, was kindly provided by Walter Schaffner, University of Zürich, Switzerland (5 originated from pGL2control (Promega). It was modified by insertion of five DNA binding sites for the yeast transcription factor Gal4 DNA-binding domain into the BglII site upstream of the strong SV40 viral promoter. The Renilla luciferase plasmid pRL-TK was obtained from Promega.tor Gal4  for use tor Gal4 . A doubltzerland . LuciferXenopus laevis A6 kidney cells . Human epitheloid cervix carcinoma cell line HeLa  was grown in DMEM, 10% fetal calf serum and antibiotics at 37° and 5% CO2. Transfections were done with commercial reagents according to the manufacturer's recommendations. HeLa and A6 cells were transfected with Fugene HD (Roche) using 3 µl transfection reagent/µg DNA while the EPC cell line was initially transfected with Fugene 6  and later on with NanoJuice .5, 0.5 µg) and Renilla  luciferase reporter plasmids that were transfected together with the to be tested effector plasmid (1.5 µg). The effector plasmids encode fusion proteins between the DNA binding domain of the yeast transcription factor Gal4  and the respective KRAB domain. The Gal4-part enables binding to Gal4 upstream binding sites in the firefly reporter plasmid. Reporter activities were measured 24 hours after transfection using the dual luciferase reagent system (Promega) as specified by the manufacturer and a Berthold single channel luminometer (Lumat LB9501). Within each experiment three replicates were done for each plasmid combination. The relative luciferase light units were normalized with the Renilla activities for each sample and the obtained values for the replicates were averaged. Fold repression was calculated by dividing the relative normalized luciferase activities of the negative control of the Gal4 DNA-binding domain alone through the activities computed for the tested effectors.Heterologous reporter cell assays were performed with cells grown in 6-well plates and using firefly g at 4°C. Aliquots from the resulting supernatant were either directly loaded to standard 10 and 12% Lämmli-type SDS polyacrylamide gels or, for concentration, first precipitated using methanol/chloroform , freshly supplemented with 1 mM DTT, Complete® EDTA-free protease inhibitors (Roche) and 1 mM sodium orthovanadate, 40 mM beta-glycerophosphate phosphatase inhibitors. Extracts were passed through QiaShredder colums and were cleared by centrifugation at 16,000 g for 10 min at 4°C. Protein concentrations in the resulting supernatants were measured by Bradford reagent . Immunoprecipitation  was performed as described previously After a brief rinse with ice-cold PBS, total sodium dodecyl sulfate (SDS) - denatured protein extracts were prepared by lysing cells with Lämmli SDS sample buffer. The extracts were sheared through QiaShredder columns (Qiagen) and centrifuged for 5 min at 16,000× loroform  and thenAfter electrophoresis, the separated proteins were electroblotted to low-fluorescence PVDF membranes  by semidry electroblotting (2 h at 25 V using BioRAD electroblotter) and immunostaining was carried out with primary antibodies and fluorescently labeled secondary antibodies at room temperature. Membranes were stained with 0.3% (w/v) Ponceau S in 3% (w/v) trifluoroacetic acid to control for even transfer. In order to avoid overbearing signals from reaction of secondary antibodies against the heavy chains of the antibodies used for immunoprecipitation, the blots were cut horizontally into different molecular weight ranges that could be processed individually. Blots were then destained in distilled water, blocked with blocking buffer  overnight, and then probed with a respective mixture of primary antibodies  for 2 hours and secondary antibodies for 1 hour , both in blocking buffer supplemented with 0.1% (w/o) Tween 20. After each antibody incubation the blots were washed 4 times 5 min each with PBS, 0.1% (w/v) Tween 20. Fluorescence signals from the blots were visualized using the Odyssey® fluorescence imager (LI-COR). Signals from IRDye800CW and IRDye680CW dyes were recorded in the green and the red channels, respectively. For representations in the figures, the original 16-bit greyscale images were loaded into Adobe Photoshop CS3 v10.0.1, and sequentially subjected to the adjustment commands “auto-contrast” and “invert” and then reduced to 8-bit. Whole blots were cropped to save space in the figures. The original 16-bit scans of the Western blots were loaded into the Odyssey application software 3.0.16 (LI-COR) and individual bands selected by manual feature selection with the rectangle tool. Signals were quantified as integrated intensity values that were corrected for local median background.Cells grown on glass coverslips were fixed in PBS 4%/w/v) paraformaldehyde, permeabilized in PBS, 0.5% (w/v) Triton X-100 and subsequently stained sequentially with primary and secondary antibodies as described in detail previously Nuclear export of Gal4 DNA binding domain fusion proteins with NES and the specified KRAB domain was assessed microscopically 24 (HeLa cells) or 48  hours after transfection after indirect immunofluorescence staining. In each independent experiment at least 100 cells per coverslip were inspected and manually scored for cells with nuclear signals brighter than cytoplasmic ones (Nuc>Cyt), about equal signal distribution (Nuc≈Cyt) or with the cytoplasmic signals being the stronger ones (Cyt>Nuc). Gal4-staining in HeLa and EPC cells was done with the polyclonal antibody from rabbit, whereas in A6 cells we had to rely on the monoclonal anti-Gal4 antibody since the one from rabbit displayed unacceptable high background in the nucleus for correct scoring.Xenopus ones using the “hmmpfam” subprogram of HMMER 2.3.1 Xenopus tropicalis genome release JGI_4.2) using the strategy described in detail by others Xenopus and are listed in Table S2. HMM matrices of the respective KRAB-A and -B subdomains were visualized as HMM-Logos http://blast.ncbi.nlm.nih.gov) were used to look for orthologs of TRIM28 in different species as indicated in the text. The lungfish sequences were kindly provided by Chris Amemiya , who did tblastn searches using the putative coelacanth TRIM28 ortholog against his local cumulative lungfish transcript database of five tissues obtained through next generation RNA sequencing Amino acid sequence alignments were made with CLC Main Workbench 6.7.2 . The alignments of the KRAB-A and KRAB-B subdomains are based on the respective amino acid sequences of ZNF10 as guide that are encoded by the KRAB-A and -B exons. KRAB domains were scored with profile hidden Markov models (HMM) of human KRAB-A and -B HMM matrices Xenopus laevis, this protein represents a member of the KRAB zinc finger protein family from the evolutionary oldest class of tetrapodes. We first cloned the XFIN KRAB domain using primers that were derived from the reference database sequence. Since the predicted N-terminus of XFIN in the database would comprise a N-terminally truncated KRAB-A subdomain we choose to extend the sequence based on the alignment to the first described member of the KRAB domain family, human ZNF10/Kox1 . Surprisingly, in-silico translation of this cloned sequence resulted in a KRAB-A amino acid sequence that aligned much better to ZNF10-A (7D8V (marked with asterisk in 24Q) and KRAB-B are identical in the former reference and the corrected XFIN KRAB domain. The XFIN KRAB-B subdomain appeared to be a relatively distant one compared to the human ZNF10-KRAB-B when scored against the human subdomain HMM  that resulted in the altered open-reading frame at the N-terminus. Thus, our data support a corrected version of the N-terminal part of the XFIN protein with a conserved KRAB-AB domain.Transcriptional repressor activities of the KRAB-AB domain of XFIN were studied in a classical heterologous luciferase reporter assay. As a derivative of the frog Figure S1A).Next, the corrected XFIN-KRAB domain was tested as fusion protein with the Gal4 DNA binding domain for transcriptional repression activity Figure 2Xenopus laevis A6 cells . Altogether the data led to the conclusion that the corrected N-terminal amino acid sequence of XFIN comprises a bona fide KRAB-AB domain in which a relatively weak KRAB-A subdomain is moderately enhanced in transcriptional repression activity by a likewise moderately potent KRAB-B subdomain.To confirm the lower extent of XFIN KRAB-B for KRAB-A potentiation we evaluated changes in transcriptional repression when KRAB-B subdomains were swapped between ZNF10 and XFIN. Compared to wildtype ZNF10-AB the ZNF10-A-XFIN-B domain chimera dropped in repression activity in Hela cells . However, these differences do not invalidate our conclusions. In summary, the N-terminus of PRDM9 that includes a subdomain similar to bone-fide KRAB-A fails to show transcriptional repression activity in a heterologous reporter gene assay.PRDM9 and the very close relative PRDM7 are the human representatives of the B domain . Their NFigure S3). Gal4-KRAB domain fusions from other KRAB zinc finger proteins confirmed the presence of telltale foci and the recruitment of endogenous TRIM28 (data not shown). In further agreement, the Gal4-KRAB fusion protein foci was colocalized with cellular HP1-alpha protein, a known interaction partner of TRIM28 Xenopus cells, since TRIM28 had not yet been described in this species and antibodies against it are not available. Yet, the existence of frog TRIM28 could be inferred from bioinformatic analyses using BLAST searches against Xenopus databases .In general, transcriptional repression activity conferred by a KRAB-domain is thought to be mediated by the TRIM28 protein that interacts through its RBCC domain with KRAB see . In a fiases see  section.While the assumption that the occurence of foci was due to interaction of the Gal4-KRAB to TRIM28 might be reasonable, colocalization does not prove physical interaction within a complex. However, if binding to endogenous nuclear TRIM28 protein would influence the localization of a Gal4-KRAB protein in an obvious manner, stable interactions might be visualized in a single cell assay. Our conceptual approach was to insert a strong nuclear export signal (NES) into all constructs immediately behind the Gal4 sequences. Provided the NES was stronger than the nuclear import signals, Gal4 should reside in the cytoplasm at equilibrium. In contrast, if a Gal4-KRAB protein interacted with endogenous nuclear TRIM28 protein, the Gal4-protein should be trapped in the nucleus, be taken out of the freely movable pool of molecules and thus display nuclear accumulation.The results of such compartmentalization assays are summarized in Figure S4A. The scoring of cells from several experiments inferred nuclear retention of the ZNF10-KRAB-AB as well as XFIN-KRAB-AB Gal4-NES fusion proteins. ZNF10-KRAB-AB appears to be slightly better in that regard. However, the difference was not statistically significant, likely because of the relative large deviations between experiments. Compared to HeLa cells, the extent of nuclear retention of the ZNF10-KRAB-AB appeared to be lower in the frog cells while that of XFIN-KRAB-AB seemed to be slightly higher. It is tempting to speculate, that the human KRAB domain from ZNF10 interacts better, i.e. with higher affinity, with its authentic human TRIM28 while XFIN-KRAB might instead bind Xenopus TRIM28 more efficiently. The different extent of retention correlated with the transcriptional reporter assay data of ZNF10 and XFIN KRAB domains.Next, the compartmentalization assay was done in A6 frog cells. Examples of the respective microscopical images of the Gal4-NES fusion proteins are given in , , Finally, we tested the stable interaction of the various KRAB domains with endogenous human TRIM28 protein in a classical co-immunoprecipitation approach. The different KRAB fusion proteins attached to GST were expressed in HeLa cells and immunoprecipitated with anti-GST antibodies. Then the eluates from the precipitation were analyzed for captured cellular TRIM28. As expected, ZNF10-KRAB-AB efficiently co-precipitated TRIM28, whereas ZNF10-KRAB-A alone or the double proline mutant of ZNF10-KRAB-AB failed to do so . When assaying the compartmentalization of the Gal4-NES fusion proteins, we observed complete cytoplasmic distribution without any clear-cut nuclear retention not only for the KRAB-PP mutant of ZNF10, but also for the ZNF10-KRAB-AB and XFIN-KRAB-AB constructs . It is tempting to speculate that the disparate transcriptional repression observed in both cellular systems could be due to fine-tuning or enhancements of factors in the mammalian lineage that have been evolved during tetrapode evolution. In addition, it will be interesting to investigate whether any other Xenopus KRAB domain might be more potent in conferring transcriptional repression in amphibian cells. Based on current gene models in the frog databases, there are KRAB-A only as well as KRAB-AB proteins. Compared to XFIN, several Xenopus proteins containing KRAB-A only as well as KRAB-B domains from AB configurations display higher HMM scores . Yet, according to these scores XFIN-AB appears to be in the upper ranks of all frog KRAB domains and a fair representative for functional tests.The fact that repression was observed in Xenopus laevis XFIN, human ZNF208 (Refseq NP_009084) has the highest score. However, reciprocal BLASTp in Xenopus resulted in other ZNF entries as top hits that do not carry KRAB domains (data not shown). The ortholog search based on sequence homology over the large phylogenetic gap between mammals and frog is hindered by the many highly conserved zinc finger sequences. A bona fide ortholog for XFIN based on sequence similarity could only be determined in Xenopus tropicalis. It is highly likely that the genome sequences encoding the NCBI XP_002942031 protein  contain the proper XFIN ortholog although the exact gene model resulting in this predicted protein might be preliminary. TRIM28 in Xenopus laevis is not well characterized. We did a BLASTp database search with human TRIM28 and chose the best hit each in Xenopus laevis (NP_001089926) and Xenopus tropicalis (NCBI XP_002937648), respectively. Reciprocal BLASTp against all human protein sequences defined TRIM28 as the human protein with highest homology and thus confirmed the two candidates as the frog orthologs. These results fit the entries in the Xenbase Xenopus database (http://www.xenbase.org). The alignments of the putative Xenopus TRIM28 proteins to human TRIM28 illustrate their conserved domain organization  tissues bona fide functional KRAB/TRIM28 module might have started to evolve around the split between lobe-finned fish and tetrapodes.We propose to define those KRAB domains as functional that show significant transcriptional repression activity in reporter assays and are able to interact with TRIM28. Based on the current state of knowledge, thus, functional KRAB domains can only be found in tetrapodes and homologs in non-tetrapodes might be considered the progeny of KRAB-like ancestor domains. The joint lack of both, functional KRAB domains and TRIM28, in ray-finned fish species are consistent with the concept that a functional KRAB/TRIM28 system of transcriptional modulation evolved in the common ancestor of the tetrapodes. Interestingly, sequences from two lobe-finned fish species, namely the recently sequenced genome of the coelacanth Figure S1Western blot analysis to compare the expression of the various Gal4 fusion protein effector constructs. Total protein extracts were made with SDS sample buffer and equal volumes of the extracts from within one experiment were subjected to Western blotting. A, B: Example blots for expression in human HeLa cells for the indicated constructs . Blots were separated in two molecular weight portions and stained with anti-GAPDH for normalization (a) or anti-Gal4 (b) to visualize the Gal4 fusion proteins. The average relative normalized Gal4/GAPDH expression values (+/− standard deviation SD) were calculated from four independent experiments using the fluorescence signals measured by the LI-COR Odyssey® fluorescence imager software. C: Experiment in Xenopus laevis A6 cells. As in A, B, with the exception that beta-actin was used for normalization purposes. * Statistical evaluation (paired 2-tailed T-test) compared the ZNF10-AB construct to the other KRAB constructs only.(PDF)Click here for additional data file.Figure S2Western blot analysis to look at the level of expression for the various Gal4 fusion proteins of the N-terminal PRDM9 portion. Total protein extracts were made with SDS sample buffer and equal volumes of the extracts. Experimental workflow and execution as described in (PDF)Click here for additional data file.Figure S3Distribution of ectopically expressed Gal4-PRDM9 N-terminal fusion proteins and colocalization analysis with endogenous TRIM28 in human HeLa cells. Cells were fixed and stained 24 hours after transfection. Each row shows the cells of the same image pane. Note, that there were no clear-cut nucleoplasmic foci in which PRDM9 domains and endogenous TRIM28 were both enriched. Bar = 10 µm.(PDF)Click here for additional data file.Figure S4Intracellular distribution of Gal4 and Gal4-NES KRAB fusion proteins 48 hours after transfection with the indicated constructs. Staining with anti-Gal4 antibodies A: Visualization by indirect immunofluorescence microscopy in Xenopus laevis A6 cells. Individual image panes a-h. Bar = 15 µm. B: Expression in Pimephales promelas EPC cells. Individual image panes a–e. Bar = 10 µm.(PDF)Click here for additional data file.Table S1DNA oligonucelotides used to construct plasmids described in the manuscript.(XLS)Click here for additional data file.Table S2List of sequences used in bioinformatic analyses. Part A: List of the presumable frog KRAB domain proteins that were used to build the HMMs of frog KRAB-A and -B. Part B: Putative TRIM28 ortholog transcripts from lungfish (Protopterus annectens).(XLS)Click here for additional data file.Table S3Quantitative data from the manual scoring and counting of the compartmentalization assay of the Gal4-NES-KRAB constructs in human HeLa, frog Xenopus laevis A6 and fish Pimephales promelas EPC cells.(XLS)Click here for additional data file.


Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown.Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain. The Eph family is the largest family of receptor tyrosine kinases, at least, 14 different Eph kinases have been identified so far. These rAdaptor proteins, composed almost entirely of well-defined interaction domains, serve to link two functional members of a signaling pathway. Nck famDrosophila and shown to be involved in LAR receptor tyrosine phosphatase-mediated motor axon guidanceSH3LKVRALKDFWNLHDPTALNVRAGDVITVLEQHPDGRWKGHIHESQRGTDRIGYFPPGIVEVVS 2ke9APWATAEYDYDAA-EDNELTFVENDKIINIEFVDDDWWLGELE----KDGSKGLFPSNYVSLGN 2k3bAPCCRGLYDFE-PENQGELGFKEGDIITLTNQIDENWYEGMIHG------ESGFFPINYVEVIV 2ew3ANative gel electrophoresis has been used to determine if the in vitro phosphorylation causes dimerization or aggregation of the SH3 domain. Protein samples were run on a 12,5% non-denaturing polyacrylamide gel. The gel was run at 50 V for 2 hrs in a 4°C cold cabinet.Abi: Abl interactor; Caskin: Cask-interacting protein; CD spectroscopy: Circular dichroism spectroscopy; GFP: Green fluorescence protein; IP: Immunoprecipitation; NIK: Nck-interacting kinase; PBS: Phosphate buffered saline; PDGF: Platelet-derived growth factor; PPII: Polyproline II helix; SAM: Sterile α motifs; SH2 domain: Src homology domain 2; SH3 domain: src homology domain 3; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; WASP: Wiskott-Aldrich syndrome protein.The authors declare that they have no competing interests.in vitro kinase assay, and measured CD spectra. AF performed the native gel electrophoresis. GB helped to maintain the cells. LB, the corresponding author, supervised the experiments and wrote the manuscript with the help of GB. All authors read and approved the final manuscript.SP, AB, and RU performed and designed the experiments. BS and AF purified the SH3 domain, carried out the Phosphomapping Results of Caskin1 and P-Caskin1.Click here for file