Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or D57NvirR onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.Toxin production in  Clostridium perfringens is a Gram-positive, endospore-forming, anaerobic pathogen that is the primary causative agent of gas gangrene or clostridial myonecrosis, and other human and animal diseases N-terminal region. Its C-terminal region contains several conserved motifs typical of histidine kinases, including the proposed site of autophosphorylation, H255, and the G box, which is thought to be involved in ATP binding N-terminal sensor region, VirS autophosphorylates at H255 N-terminal region of VirR. This region also contains the E8, D9 and K105 residues, which in other response regulators are highly conserved and form a phosphoacceptor pocket C-terminal DNA binding domain pfoA, by binding independently to two imperfect 12-bp directly repeated sequences, called VirR boxes, located upstream of the pfoA promoter C. perfringens genomes The VirS sensor histidine kinase is predicted to contain six or seven transmembrane domains in its N-terminal region(s) required for VirS function have not been determined and the role of phosphorylation in transcriptional activation has not been demonstrated. In this paper we identify a region located within one of the putative transmembrane domains that appears to be required for VirS function and show, using in vitro phosphorylation studies, that a truncated form of VirS is able to undergo autophosphorylation and subsequently act as the phosphodonor for VirR. Using perfringolysin O production as a reporter system for VirR function, we showed that activation of transcription was dependent on the presence of VirS and the D57 residue of VirR. These studies demonstrated that phosphotransfer from VirS to VirR and gene dosage were important factors in the regulation of perfringolysin O production by VirR.Although the role of the VirSR system in toxin production has been established, the Escherichia coli strains were grown at 37°C in 2×YT or SOC media −1 ampicillin or 30 µg ml−1 chloramphenicol. C. perfringens strains were grown at 37°C in fluid thioglycollate medium (FTG) (Difco), trypticase-peptone-glucose (TPG) broth −1 erythromycin (NAEm50) or 30 µg ml−1 chloramphenicol (NACm30) was used for selection purposes. To screen for phospholipase C production, C. perfringens transformants were grown on egg yolk agar −1 chloramphenicol (EYACm30), whilst perfringolysin O activity was visualised on horse blood agar (HBA) C. perfringens agar cultures were incubated under anaerobic conditions in 10% (v/v) H2, 10% (v/v) CO2 in N2.Bacterial strains are listed in The level of perfringolysin O activity in the culture supernatants was determined by a doubling dilution hemolysin assay using horse red blood cells, as previously described E. coliC. perfringensPlasmid DNA was isolated from E. coliC. perfringensC. perfringens genomic DNA was isolated from 5 ml FTG broth cultures as before Competent virS mutants were isolated after passage through the DNA repair deficient strain Epicurean Coli® XL1-Red (Stratagene). The target plasmid was pJIR884 virS gene and the upstream virR promoter. Four independently derived pJIR884 DNA samples were obtained from transformed XL1-Red cells and used to transform C. perfringens strain JIR4000, a JIR325 derivative in which the virS gene has been insertionally inactivated by Tn916virS mutants were detected as non-hemolytic colonies on HBA supplemented with 50 µg ml−1 erythromycin. Since perfringolysin O production is dependent upon the VirSR system virS mutation in JIR4000, i.e. no hemolysis on HBA, was used as a direct means of detecting loss of VirS function. To facilitate analysis, plasmid DNA was recovered from all non-hemolytic strains of C. perfringens and used to transform E. coli. Sequence analysis was carried out on DNA isolated from the resultant E. coli strains to identify plasmids with mutations within the virS gene. Sequence analysis was used to identify plasmids with mutations within the virS gene. Selected plasmids derived from both methods of random mutagenesis were further analysed and are listed in Random virS gene carried on pJIR2056 was performed using a modification of the unique site elimination method in vitro Site-Directed Mutagenesis Kit (Promega) virS genes and upstream promoter regions of the mutated plasmids were sequenced to confirm that only the desired mutation was present. To test the effect of the mutations on VirS function, a 3.8-kb XbaI/SalI fragment from each mutated plasmid was isolated, cloned into the XbaI/SalI site of the E. coli/C. perfringens shuttle vector pJIR751, and used to transform C. perfringens strain JIR4000. Site-directed mutagenesis of the virR gene and the cloning of the resultant mutated genes were carried out as described previously Site-directed mutagenesis of the H255I and VirScG402D, a 0.69 kb PCR product was generated using the primers JRP1873 and JRP1133  (Novagen) to construct pJIR2699, pJIR2792 and pJIR2825, respectively  H255I & VirScG402D) or 1 h at 37°C (VirR & VirRD57N). Proteins were purified using Talon resin (Clonetech) and dialysed as described previously To induce the expression of recombinant proteins, 2, 0.5 mM EDTA, 2 mM DTT) to a final concentration of 20 µM, and aliquoted for use in individual phosphorylation reactions. Phosphorylation was initiated by the addition of 0.1 volumes of a 10× reaction mixture containing either 2.5 µM [γ-32P]ATP (111TBq/millimole) (Perkin Elmer Life Sciences) and 247.5 µM unlabelled ATP, or 247.5 µM unlabelled ATP alone. The latter reaction mixture was added to unlabelled reactions that were subject to Western blot analysis using a Penta-His antibody (Qiagen). To examine autophosphorylation specificity, reactions were pre-incubated with 50 mM EDTA or 2.5 mM unlabelled ATP at room temperature (approx 23°C) for 5 min before the addition of the 10× reaction mixture. All reactions were incubated at room temperature for 5 min, stopped by the addition of an equal volume of 2× gel loading buffer, and then subjected to SDS-PAGE VirSc was diluted in phosphorylation buffer  and incubated at room temperature for 1 h. VirR or VirRD57N was then added to a final concentration of 0.9 µM, and reactions were incubated for a further 15 min at room temperature before the addition of 10× reaction mixture and incubation at room temperature for varying amounts of time. Proteins were separated by SDS-PAGE and gels were treated as before. The presence of proteins in these phosphorylation studies was verified by Western blot analysis using His-tag antibodies on duplicate reaction samples (data not shown).For phosphotransfer studies, VirSc (10 µM) was autophosphorylated as above, in a total volume of 10 µl, with the exception that reactions were carried out in phosphotransfer buffer  was added and the cells were lysed by vigorous agitation, twice for 45 sec, in a FastPrep FP120 Cell Disrupter (Savant/Bio101). Cell debris was removed by centrifugation at 12,000 g for 10 min at 4°C, and the soluble whole cell extracts were collected. Following determination of the protein concentration, 20 µg of each extract was used in Western blot analysis with VirR-specific antisera.Following SDS-PAGE, proteins were transferred to Protran nitrocellulose membrane (Schleicher and Schuell) using a mini Trans-Blot® Electrophoretic Transfer Cell (BioRad) for Western blot analysis. The nitrocellulose membrane was developed using Penta-His antibody (1∶2000 dilution) or VirR-specific antiserum (1∶1000 dilution) virR and D57NvirR onto the chromosome, the genes were PCR amplified using primers JRP2812 and JRP2813 (MluI sites that facilitated cloning into the unique MluI site within the plc target region of pJIR750ai virR) and pJIR3243 (D57NvirR) were introduced into C. perfringens strain TS133 .To construct the plasmids used to introduce  JRP2813 . The oliin TS133  by elect JRP2874 . To cureC. perfringens total RNA was isolated as described previously rpoA gene for each strain, and the results expressed as a proportion of the wild-type.C-terminal domains of sensor histidine kinases, including the putative site of autophosphorylation, H255, and the G (GxGL) motif , Phobius (http://phobius.cbr.su.se/) and Sosui (http://bp.nuap.nagoya-u.ac.jp/sosui/). These algorithms showed a consensus topology, where the extracellular N-terminus was followed by seven transmembrane domains, and the catalytic C-terminal region was cytoplasmically located  motif . In prev located . AttemptN-terminal domain. Mutagenesis of the +virS shuttle plasmid pJIR884 virS structural gene, 10 of which were unique (virS genes were still transcribed in C. perfringens (data not shown).With this model in mind, random mutagenesis was used to identify VirS residues of functional importance, particularly within the e unique  and there unique , indicatN-terminal region, within a putative transmembrane domain (TMD 4). The other changes were all located within the cytoplasmic C-terminal domain. Four substitutions, H255Y, D256G, N259K and H260Y, were located either in or near H255, the putative site of autophosphorylation. One alteration, C335Y, was close to the conserved N box, a region predicted to form part of the catalytic domain that binds ATP. The G402D and G415R substitutions were located in or near the G box, which is also predicted to form part of the catalytic domain Three of the VirS substitutions, L99F, T100A and E102K, were located in the N-terminal portion of VirS, several leucine residues on either side of L99, and upstream of E102, were targeted for substitution. The sequence of this leucine rich region is 88IMISLIFWLFMLTVEAL104. The leucine residues adjacent to L99, L104 and L96, were altered to phenylalanine, on the basis that the random L99F mutation eliminated VirS function. The L104F substitution had no significant effect on perfringolysin O production  was cloned into the pET-22b(+) expression vector, and a 27.7 kDa 6×-His tagged protein (VirSc) overexpressed. After purification on a Talon column, VirSc was used in autophosphorylation experiments using [γ−32P]ATP or [α-32P]ATP. A single band corresponding to the correct size of VirSc (27.7 kDa) was observed in the [γ−32P]ATP reaction , or the first glycine residue in the conserved G box (G400A), was shown to eliminate VirS function rScH255I , lane 2,horylate , lane 3.e  was tested under the same conditions (D57N was not able to be phosphorylated and that D57 was most likely the site of phosphorylation in VirR.Using phosphorylated VirSc (VirSc-P) as the phosphodonor, we then tested the ability of purified VirR to act as a phosphoacceptor. The appearance of a labelled band corresponding to VirR was observed . This renditions . FurtherD57N protein was functional in vivo, the D57NvirR gene was subcloned into the E. coli-C. perfringens shuttle vector, pJIR750 C. perfringens virR mutant TS133 virR mutation in TS133 with a plasmid carrying a wild-type virR gene results in the restoration of perfringolysin O activity D57NvirR gene was introduced into TS133, the resultant strain carrying the mutated gene produced less perfringolysin O than the wild-type, but still produced very significant levels of the toxin -targeted Targetron vector virR genes back onto the chromosome in single copy. Using this method we constructed separate strains in which the wild-type virR gene or the D57NvirR gene was inserted into the plc gene. As a result of the genetic organisation of the virRS operon, the original insertional inactivation of virR in TS133 also eliminated the expression of virSvirS gene virS in trans. Control strains carried the vector plasmid pJIR751.We postulated that this apparent phosphorylation independence occurred as a result of gene dosage effects. To test this hypothesis, we utilised an α-toxin  resulted in wild-type levels of perfringolysin O activity Click here for additional data file.Table S2(0.06 MB DOC)Click here for additional data file.


In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control. The Ubiquitin-Proteasome System regulates protein degradation via several steps. The cullin-RING E3 ligase machinery is involved in one of these. In this step, ubiquitin is transferred from E2 to the substrate protein, labeling the substrate protein for degradation. However, when E3, E3-substrate and E2-ubiquitin crystal structures are modeled together, the distance between ubiquitinated E2 and the substrate binding site is ∼50–59Å, raising the question how the E3 machinery bridges the distance and orients the substrate for the ubiquitin transfer. We performed explicit solvent simulations for all nine available substrate binding protein complexes in the PDB, with and without the corresponding E3 components to which they are bound. In all of these nine substrate binding proteins, we noticed a flexible linker that rotates the substrate binding domain to a great extent in the same direction, toward the E2-ubiquin. We further noticed that the flexibility is regulated allosterically by binding events associated with either domain. The results suggest that the flexible linker serves as a hinge to rotate the substrate binding domain and to accurately position the substrate for ubiquitination. As such, the simulations suggest an answer to the question of how the machinery operates to orient the substrate for ubiquitination. The Ubiquitin-Proteasome System (UPS) regulates protein degradation in many cellular processes, including signaling, cell-cycle control and development The ubiquitin system cascade is pyramidal, allowing efficiency and specificity. A single E1 transfers ubiquitin to dozens of E2, which together with hundreds of E3 ubiquitinate thousands of substrates Skp2  – Rbx - E2 complex by superimposing the Cul1-Rbx1-Skp1-F box on the Skp1-Skp2 complex Zheng et al Skp2 structure is also believed to be rigid in vivo; this suggests that the Cdc4 linker is critical for the Cdc4 function Yet, the lack of flexible linkages in the cullin scaffold is an intrinsic common feature for E3 substrate-binding proteins. The linker serves as a hinge to orient the substrate, optimally positioning it for the ubiquitin transfer from E2. This feature could also facilitate the favored orientation during the ubiquitin transfer process in multi-ubiquitin labeling and/or substrate dissociation from the E3 ligase. To test our hypothesis, we performed molecular dynamics simulations for nine substrate binding proteins whose crystal structures are available, including F-box proteins Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, and TIR1; VHL-box protein pVHL; and SOCS-box proteins SOCS2 and SOCS4, in unbound and bound forms. For all nine simulated proteins, the inter-domain linker regions were flexible in the unbound form, moving away from the E2; while in the bound form, the flexibilities significantly decreased, yet still moving toward E2. We investigated the driving forces and noticed that hydrophobic core formation and charge-charge interaction appear to play an important role. More interestingly, we observed the presence of a conserved proline in the linker region. While there is no cis/trans conformational switch, the prolines demonstrate substantial pucker in the unbound form, and the puckering is significantly decreased in the bound form. We further noticed that the proline is at the hinge and in all nine proteins the puckering is coupled with the backbone conformational change. This observation suggests that this conserved proline has a role in the conformational change in the unbound form and constrains the conformation in the bound form. We propose that intrinsic linker flexibility is a common feature in substrate binding proteins, optimally positioning and orienting the substrate for ubiquitin transfer in the E3 ligase system. Following accurate geometrical positioning for the transfer, the linker flexibility is reduced, moving it further toward optimal conformational orientation.These observations led us to hypothesize that the flexibility of the inter-domain linkers of substrate-binding proteins VHL-box protein pVHL and SOCS-box proteins SOCS2 and SOCS4 have two domains, the box and the substrate binding domains. The structures of the conserved C-terminal box domains, VHL-box and SOCS-box, respectively, consist of three α helices, H1, H2 and H3; both VHL-box and SOCS-box domains interact with Elongin C. On the other hand, the N-terminal substrate binding domains, β domain for pVHL and SH2 domain for SOCS2 and SOCS4, are very different. Yet, when we superimpose the Elongin C and box domain of pVHL and SOCS2, the distance between the hydroxylation site (Hyp564) of pVHL substrate HIF-1α Similar to VHL-box and SOCS-box proteins, F-box proteins also have two domains, the F-box and the substrate binding domains. F-box domains are structurally conserved, while the substrate binding domains are not. The substrate binding domains of Skp2 and TIR1 have leucine-rich repeats; Fbs1 has sugar binding domain; whilst those of Fbw7, β-TrCP1 and Cdc4 have a WD-40 substrate binding domain. Nonetheless, superimposing Skp1 and ASK1 of the Skp1/F-box and ASK1/F-box of the six F-box complexes as an anchor, leads to an interesting result: four of these proteins, Skp2, Fbw7, Cdc4 and Fbs1 overlap the sites that the substrates bind: pThr187 of Skp2 substrate p27 To our knowledge, complexed crystal structures are available for five Skp1-binding F-box proteins, Skp2, Fbw7, β-TrCP1, Cdc4, and Fbs1; one ASK1-binding F-box protein TIR1, and three Elongin C- binding proteins, VHL-box protein pVHL, and two SOCS-box proteins, SOCS2 and SOCS4. Molecular dynamics simulations were performed for all nine proteins in the unbound and bound  forms. To decrease the chances of the results dependence on the starting conditions, repeated simulations were performed for all the unbound forms. The rotation angles for both unbound (including two independent simulations) and bound forms are shown in For the unbound form, when the conserved box domains are superimposed, the substrate binding domains rotate up to 30–80 degrees with respect to their corresponding box domains in the 20 nanosecond simulations. In the bound form simulations the rotations have significantly decreased with the substrate binding domain still moving further toward E2-ubiquitin. Compared to the unbound form, the maximum and the average rotation angles are much smaller: the maximum angles of the F-box proteins Skp2, Fbw7, β-TrCP1 and Cdc4 are 27.9, 32.8, 40.8 and 27.0 degrees, respectively , decreasWe searched for the driving forces for the conformational change. The inter-domain interface structures of F-box proteins are quite different than those of the VHL-box and SOCS-box proteins. The three helix bundle and interface of F-box proteins form a cavity. In the unbound form, they tend to form a hydrophobic core consisting of H2 of the F-box and an α helix  next to the substrate binding domain. Hydrophobic core formation can assist in driving the conformational change in F-box proteins. The distance changes between the hydrophobic residues of F-box proteins during the simulations are shown in For the SOCS-box proteins SOCS2 and SOCS4, charge-charge interactions could also play a role in inter-domain rotation. SOCS2 has two positively charged residues at the interface, Arg41 and Arg168. During the simulations, these two charged residues separated from 4.6 Å to more than 20 Å . Even thThe rotation hinges are in the linker region for all nine proteins. For TIR1, the LRR1 serves as its linker region. The VHL-box and SOCS-box proteins have only one hinge region in the short linker between the two domains, while the F-box proteins have two hinge regions: the first is in the short turn next to the F-box; the second in the α helix next to the substrate binding domain. Specifically, the second hinge is at the beginning of the α helix of Skp2, Fbw7 and β-TrCP1, Fbs1, but at the end of the α helix of Cdc4 and TIR1. Sequence analysis of the hinge region  shows onCovariance maps were generated for the nine proteins for the unbound and bound form simulations. Covariance maps are useful in identifying regions whose motions are correlated or anti-correlated and as such can assist in discovering allosterically-related residues Here we investigate a crucial mechanistic detail of the E3 ligase system: in order for ubiquitin to be efficiently transferred to its cognate substrates, the substrates have to be precisely spatially positioned and oriented with respect to the E2-ubiquitin . Yet, cuIn a recent conformational study of ubiquitin Further, superposition of the two Elongin C complexes, with pVHL and with SOCS2, indicates that the respective substrate binding sites overlap even though their substrate binding domains are structurally dissimilar; while in SOCS4 the position is different. Yet, the necessity to raise the temperature to 340 degrees in the SOCS4 simulations to observe the hinge motion and re-orientation  suggestsHydrophobic core formation for F-box proteins and charge-charge interactions for VHL-box and SOCS-box proteins could be the driving forces for the conformational change of the linker region. We further noticed a conserved proline in the linkers of all nine proteins. Conformational analysis indicated a large difference in the proline pucker between the bound and unbound simulations. Proline ring puckering has been coupled to the backbone conformation change Covariance maps also show strong correlations between the linker and the two domains. The coupled motion between the substrate binding domain and the linker implies that substrate binding can allosterically affect the linker conformation. These results are consistent with recent hydrogen exchange mass spectrometry showing that Skp2 substrate-binding domain binding to Cks1 causes a conformation change of the Skp2 linker To conclude, The starting structures of the unbound and bound forms of nine proteins, Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4 were obtained from crystal structures . The starting structures of the bound forms were abstracted with substrate removed, except SOCS2 and SOCS4, whose substrates were not in crystal structure, whereas the starting structures of the unbound form were created by removing all the binding partners, including the adaptor and the substrate, if applicable. For the pVHL bound to Elongin C, only Elongin C residues 58–112 were used since coordinates were unavailable for the eight-residue gap between residues 49 and 58, and the solved Elongin C structure for residues 17 to 49 was far away from the pVHL-Elongin C binding site. The starting structure of SOCS2/SOCS4 -Elongin C complexes was generated with similar Elongin C truncation. All models were solvated in a TIP3P water box with a minimum distance of 10 Å from the edge of the box to any protein atom. The system charges were neutralized by adding chloride or sodium ions.Molecular dynamics (MD) simulations were performed with CHARMM 27 Structural alignments and figure rendering were performed by VMD. The angle rotations at 0 and 20 ns were calculated by DynDom Figure S1Angle rotation graphs of unbound trajectory 1 (black), trajectory 2 (blue) and bound (red) forms for (A) Skp2, (C) Fbw7, (E) β-TrCP1, (G) Cdc4, (I) Fbs1, and (K) TIR1. The graphs of changes in the distances between hydrophobic residues from box domain and linker are shown for the unbound form trajectory1 of (B) Skp2, (D) Fbw7, (F) β-TrCP1, (H) Cdc4, (J) Fbs1 and (L) TIR1.(1.82 MB PDF)Click here for additional data file.Figure S2Angle rotation graphs of unbound trajectory 1 (black), trajectory 2 (blue) and bound (red) form for (A) pVHL, (C) SOCS2, (E) SOCS4. The graphs of distance changes between the charged residues at the inter-domain interface are shown for the unbound form trajectory1 of (B) pVHL, (D) SOCS2, (F) SOCS4.(0.98 MB PDF)Click here for additional data file.Figure S3Models of the E2-Rbx1-Cul1-Skp1 complex superimposed with (A) Fbw7 (B) β-TrCP1 (C) Cdc4(D) Fbs1 and (E) TIR1. E2 (purple) is docked to Rbx1(gray)-Cul1(blue)-Skp1(red)-Skp2 F-box (yellow) complex (PDB code 1LDK). Snapshots of (A) Fbw7 (B) β-TrCP1 (C) Cdc4 (D) Fbs1 and (E) TIR1 at 0 ns (orange) and 20 ns (green) for unbound form and 20 ns (cyan) for bound form are superimposed with Skp2 F-box domain.(1.04 MB PDF)Click here for additional data file.Figure S4Covariance maps of (i) unbound and (ii) bound form of (A) Skp2 (B) Fbs1 (C) TIR1 (D) Fbw7 (E) β-TrCP1 and (F) Cdc4. The position of the prolineis marked. The more red, the stronger the positive correlation; the more blue the stronger the negative (anti-) correlation. The bar provides the scale.(2.50 MB PDF)Click here for additional data file.Figure S5Covariance maps of (i) unbound and (ii) bound form of (A) pVHL(B) SOCS2 and (C) SOCS4. The position of the prolineis marked. The more red, the stronger the positive correlation; the more blue the stronger the negative (anti-) correlation. The bar provides the scale.(0.72 MB PDF)Click here for additional data file.Figure S6Sequence alignment of (A) VHL-box, SOCS-box and (B) F-box proteins.(0.10 MB PDF)Click here for additional data file.Table S1Rotation angles (degrees) for nineproteins.(0.12 MB PDF)Click here for additional data file.Table S2Sequence analysis of the conserved prolines. The sequences of box-domain and linker region for each protein were used as query sequences to search for matching sequences using BLAST.(0.09 MB PDF)Click here for additional data file.


A combination of computational and experimental approaches reveals the dynamics of ASIC1 gating, involving a deformation of the channel that triggers “twist-to-open” motions of the channel pore. The acid-sensing ion channel 1 (ASIC1) is a key receptor for extracellular protons. Although numerous structural and functional studies have been performed on this channel, the structural dynamics underlying the gating mechanism remains unknown. We used normal mode analysis, mutagenesis, and electrophysiological methods to explore the relationship between the inherent dynamics of ASIC1 and its gating mechanism. Here we show that a series of collective motions among the domains and subdomains of ASIC1 correlate with its acid-sensing function. The normal mode analysis result reveals that the intrinsic rotation of the extracellular domain and the collective motions between the thumb and finger induced by proton binding drive the receptor to experience a deformation from the extracellular domain to the transmembrane domain, triggering the channel pore to undergo “twist-to-open” motions. The movements in the transmembrane domain indicate that the likely position of the channel gate is around Leu440. These motion modes are compatible with a wide body of our complementary mutations and electrophysiological data. This study provides the dynamic fundamentals of ASIC1 gating. The acid-sensing ion channels (ASICs) are key receptors for extracellular protons and are becoming increasingly important drug targets. However, their gating mechanism is still not fully understood. The crystallographic structure of the ASIC1 protein provides a clue, but the dynamics of the channel remains to be elucidated. Using computational biology, site-directed mutagenesis, and electrophysiological recordings, we investigated the dynamics of ASIC1 gating. Through “normal mode analysis,” we detected a series of collective motions between the beta turn and transmembrane domain, and between the thumb and finger domains, suggesting a deformation pathway related to channel gating. The intrinsic rotation of the extracellular domain and the collective motions between the thumb and finger domains that are induced by proton binding serve to deform the channel from the extracellular to the transmembrane domain, triggering a “twist-to-open” motion of the channel pore. The relationship between the dynamics and the gating mechanism was experimentally confirmed by a series of complementary mutations in ASIC1 and electrophysiological measurements. Our study also indicated that the likely position of the channel gate is around Leu440 within the ASIC1 protein. We propose a clear model correlating the structural dynamics of ASIC1 and its gating mechanism. Each subunit is composed of two domains, a large extracellular (EC) domain, and a transmembrane (TM) domain. The EC domain resembles a clenched hand, which can be further divided into finger, thumb, palm, knuckle, and β-turn subdomains. The TM domain comprises two transmembrane helices, TM1 and TM2, in a “forearm” arrangement  channe1 Computational simulation has been a promising tool to address the dynamic behavior of biological molecules. Recently, Shaikh and Tajkhorshid carried out molecular dynamics (MD) simulations on cASIC1, which provided useful information for the potential binding sites of cations and protons in ASIC1 In the present study, the dynamic behavior of ASIC1 has been studied on the basis of the crystal structure of cASIC1 using NMA along with complementary mutagenesis and electrophysiological experiments. The NMA revealed complementary twisting motions throughout the receptor, with which the ion channel may undergo an open motion. Further analyses on the motion modes detect a series of collective movements among the subdomains of EC domain, which control and induce the motions of channel pore. Furthermore, the twisting motion modes of the TM domain indicate the probable position of the channel gate. Electrophysiological assays on the human ASIC1a (hASIC1a) mutants of a series of key residues associated with the motions support the computational results. This study provides new information on the intrinsic dynamic behavior of ASIC1 motions associated with the channel opening, enabling us to construct a new model for the gating mechanism of the channel. This model, supported by a number of key experimental observations from others as well as our own, for the first time to our knowledge, provides a clear picture of the correlation between the structural dynamics of ASIC1 and its gating mechanism.NMA is a computational approach that can efficiently predict the collective dynamics and inherent flexibilities in biological macromolecules. Accordingly, we used NMA to detect the intrinsic motion modes of ASIC1. First, we examined the low-frequency modes of ASIC1 produced by NMA because they may reflect the global motions of the ASIC1 channel and are often potentially related to biological functions NMA revealed that the most relevant modes of ASIC1 to its gating mechanism might be modes 1 and 3, which undergo similar motions. In addition to the rock and rotation of the EC domain , the six+, Li+, and K+ permeability of these mutated channels were also significantly shifted (+/K+ selectivity) with respect to the WT channel under standard intra- and extracellular ion compositions  of Q437N mutant decreased significantly  for both L440A and A444G mutants decreased dramatically , we constructed eight mutants around the putative gate position of ring-I and ring-II : G436A,  shifted  and S4. ositions , the selficantly . Furtherficantly . These ratically . We attrIt should be emphasized that the identified key residues contributing to the possible gate of ASIC1 are mostly conserved among the superfamily of ASICs as indicated by the sequence alignment . On the After detecting the motions of the pore and the probable gate position of the channel, we asked how the channel pore undergoes the twisting-to-open motion. For this purpose, we examined the relationship between conformational changes of other parts of the EC and the TM domain. Structurally, the β1 and β12 strands are connected to TM1 and TM2, respectively; the β9 and β10 strands are linked to the thumb subdomain; β1, β12, β9, and β10 form a metacarpal plane (M-plane) . There iKey interactions between the β turn and the TM domain important for the gating mechanism were further characterized by electrophysiological experiments. To this end, we designed mutations to respectively disrupt the π–π stacking interaction between Y72 and W288, the C-H···π hydrogen bonding The C-H···π hydrogen bond between P287 and Y72 represents another important interaction that may be responsible for the collective motion of the β turn with the TM domain. In addition, because P287 is located at the lip of the β turn, it may play a structural role in stabilizing the β-turn conformation and likely affect channel gating. As predicted, the P287G mutation abolished the channel opening capacity . On the Of note, the importance of the interaction between Y72 and W288 to the channel gating was also addressed by Li et al. 50  of the acid-induced currents  calculations indicate that all these mutations decrease the binding affinity between the thumb and finger  are larger than 0.9  and in the trimer revealed the most flexible portions of cASIC1. Each subunit is more flexible in the isolation state than in the trimer, suggesting that the trimerization of the receptor restrains the mobility and plasticity of the subunits . Still, We investigated the holistic motions of the EC domain by analyzing the motion modes. The EC domain as an entire body may undergo rotation around the pore axis and swing round the wrist region of the receptor. For example, the EC domain in mode 2 displays a rocking vibration , and in Two important findings that were significant to the gating mechanism were uncovered amongst the normal modes of EC domain. The first is the consistent motions between thumb and finger domains . This reNMA also revealed a notable twisting to open pore motion located in the TM domain, which is directly associated with channel gating . Most im+ ion may bind to an acidic pocket between thumb and finger subdomains. This process provides the energy to trigger movement of the EC domains; however, how the H+ binding would drive such events is still unclear. Here, we provide a possible explanation based on our NMA and mutagenesis experiments.The function of the ASIC large EC domain in the gating mechanism remains to be elucidated. Results of this study indicate that motions of all subdomains and regions of the EC domain may collectively stimulate the motions and conformational changes of the TM domain, affecting the shape of the channel pore . The dyn+ binding to the acidic pocket enhances the interaction between these two domains, which heightens their intrinsic motion during gating process. This hypothesis has been verified by a series of mutations and electrophysiological determinations: mutations that either abolish the H-bonds or weaken the hydrophobic interaction between these two subdomains shift the dose–response curve to a lower pH region and decrease the pH50 values; and the mutations of D238K and D238S that might enhance hydrogen bonding between the thumb and finger shift the dose–response curve to the higher pH region and increase the pH50  http://lorentz.immstr.pasteur.fr/nomad-ref.php) dij is the distance between two atoms i and j, c is the spring constant of the Hookean potential , and Rc is an arbitrary cut-off. In this study, Rc was set to be 10 Å.The atomic coordinates for the crystal structure of cASIC1  of atomic motion were computed with the modes and frequencies derived from the NMA using Equations 3 and 4 mi and mj are the masses for atoms i and j; ωk is the vibration frequency of mode k; aik and ajk are the ith and jth components of the kth eigenvector.Cross-correlations (All constructs were expressed in CHO cells. Transient transfection of CHO cells was carried out using Lipofectamine 2000 (Invitrogen). Electrophysiological measurements were performed 24–48 h after transfection. The cDNA encoding hASIC1a was a generous gift from Jun Xia .3, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose, aerated with 95% O2/5% CO2 . The standard external solution contained (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, and 10 glucose, buffered to various pH values with either 10 mM HEPES (pH 6.0–7.4), or 10 mM MES (pH<6.0). The standard patch pipette solution for whole-cell patch recording was (in mM): 120 KCl, 30 NaCl, 1 MgCl2, 0.5 CaCl2, 5 EGTA, 2 Mg-ATP, 10 HEPES. The internal solution was adjusted to pH 7.2 with Tris-base. Unless otherwise noted, the electrophysiological recordings were carried out under standard conditions.The incubation solution contained the following components (in mM): 124 NaCl, 24 NaHCO+, K+, and Na+, the internal solution contained (in mM): 150 NaCl, 10 EGTA, and 10 HEPES, and the external solution contained (in mM): 150 test monovalent cation (X), 10 HEPES (replaced with MES when pH is 5.0), 10 glucose, and 2 CaCl2. The relative permeability of Li+ and K+ over Na+ was measured by comparing the reversal potentials when the external solution contained LiCl, KCl, or NaCl with internal NaCl in each case. The osmolarities of all these solutions were maintained at 300–325 mOsm (Advanced Instruments).For measurement of the relative permeability of LiSolutions with different pH values were applied using a rapid application technique termed the “Y-tube” method throughout the experiments The human ASIC1a cDNA was subcloned into the pEGFPC3 vector (Promega Corporation). Each mutant was generated with the QuikChange mutagenesis kit (Stratagene) in accordance with the manufacturer's protocol.The electrophysiological recordings were performed in the conventional whole-cell patch recording configuration under voltage clamp conditions. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller . The resistance between the recording electrode filled with pipette solution and the reference electrode was 4–6 MΩ. Membrane currents or potentials were measured using a patch clamp amplifier  and were sampled and analyzed using a Digidata 1320A interface and a computer with the Clampex and Clampfit software . In most experiments, 70%–90% series resistance was compensated. Unless otherwise noted, the membrane potential was held at −60 mV throughout the experiment under voltage clamp conditions. All the experiments were carried out at room temperature (22–25°C).t-test. The permeability ratios of pNa/pLi and pNa/pK were determined by the modified Goldmann-Holdgkin-Katz equation: pX/pNa = exp (ΔVrevF/RT) due to the equimolar cations in the external and internal solution, where X represents the test cation, ΔVrev is the change in reversal potential when Na+ was replaced by the tested cation, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.Results were expressed as the mean±SEM. Statistical comparisons were made with the Student's Figure S1The motions correlation map of residues in subunit A. Correlated motion maps are represented with a color code related to the sign and intensity of correlations (ranging from dark blue for noncorrelations to dark red for perfect correlations). The color bar on the top indicates the regions of the TM, palm, thumb, and finger domains.(4.35 MB TIF)Click here for additional data file.Figure S2Bending and swing vibrations between finger and knuckle detected by NMA. The motions in modes 8, 12, and 16 are shown as examples. The vector arrows represent the amplitude and direction of the displacement experienced by each residue of these two subdomains. The arrows clearly illustrate the bending and swing vibration between them.(4.30 MB TIF)Click here for additional data file.Figure S3Representative traces and magnitudes of the inward currents for ASIC1 mutants. (A) Representative traces of the inward currents from CHO cells transfected with WT or mutated ASIC1. (B) Collected data exemplified in (A).(10.47 MB TIF)Click here for additional data file.Figure S4Typical examples of I/V relationship curve under different conditions. Channels were activated with pH 5.0 in the whole cells recording. Once currents were fully activated, a fast (800 ms) voltage ramp was run from −90 mV to +90 mV and reverse potential was determined. The application pipette contained (in mM): either 150 NaCl (Naout), 150 LiCl (Liout), or 150 KCl (Kout) and 10 glucose, 10 HEPES, and 2 CaCl2. The patch pipette contained (in mM): 150 NaCl, 10 HEPES, and 5 EGTA (Nain).(8.66 MB TIF)Click here for additional data file.Table S1Reversal potentials obtained from CHO cells transfected with WT or mutated ASIC1 channel under different conditions.(0.04 MB DOC)Click here for additional data file.Table S2Calculated binding free energy (ΔGbinding) between the thumb and finger subdomains of the WT channel and its mutants.(0.03 MB DOC)Click here for additional data file.Text S1Supplemental experimental procedure.(0.04 MB DOC)Click here for additional data file.Video S1The shearing vibration of the M-plane in mode 3 induces TM1 and TM2 helices to undergo twisting movements.(0.45 MB AVI)Click here for additional data file.Video S2The bending of the M-plane in mode 11 triggers a swinging motion to the TM domain.(0.45 MB AVI)Click here for additional data file.Video S3Bending and swing vibrations between finger and knuckle evoke motions of the TM domain through thumb and M-plane in mode 8. The subunit is shown in ribbon representation with subdomains colored in green (thumb), magenta (finger), cyan , orange (knuckle), blue (β turn), and red (TM).(0.47 MB AVI)Click here for additional data file.Video S4Bending and swing vibrations between finger and knuckle evoke motions of the TM domain through thumb and M-plane in mode 12. The subunit is shown as (0.49 MB AVI)Click here for additional data file.Video S5Bending and swing vibrations between finger and knuckle evoke motions of the TM domain through thumb and M-plane in mode 16. The subunit is shown as (0.48 MB AVI)Click here for additional data file.


RIZ encodes two different protein products, RIZ1 and RIZ2, which have been identified to be a tumor suppressor and a proto-oncoprotein, respectively. Structurally, the two protein products share the same amino acid sequences except that RIZ2 lacks an N-terminal PR domain with methyltransferase activity. Previous studies have shown that over-expression of RIZ2 is usually associated with depressed RIZ1 expression in different human cancers. It is generally believed that RIZ1 and RIZ2 regulate normal cell division and function using a “Yin-Yang” fashion and the PR domain is responsible for the tumor suppressing activity of RIZ1. In order to better understand the biological functions of the PR domain by determining its three-dimensional crystal structure, we expressed, purified and crystallized a construct of the PR domain (amino acid residues 13–190) in this study. The maximum size of the needle-shaped crystals was approximately 0.20 × 0.01 × 0.01 mm.Through alternative promoter usage, human retinoblastoma protein-interacting zinc finger gene  RIZ, was first identified from a functional screening for retinoblastoma tumor suppressor binding genes . Needle-shaped crystals were obtained within 5 days from drops containing equal-volume mixtures (2 μL:2 μL) of protein solution  and reservoir solution  equilibrated against the reservoir solution (0.6 mL). The crystals were confirmed to be the PR domain by staining with the IZIT Crystal Dye™ from Hampton Research .In the current study, the PR domain (amino acid residues 13–190) of human tumor suppressor RIZ1 was cloned and over-expressed. The recombinant PR domain was confirmed to be active by the methylation reaction and crystallized by the sparse-matrix protocol. The maximum size of the needle-shaped crystals was 0.20 × 0.01 × 0.1 mm. Optimization of the initial crystallization conditions to obtain large crystals suitable for X-ray diffraction is in progress.


Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX  experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.We determined the crystal structure of the SBDS orthologue from It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function. Methanothermobacter thermautotrophicus mth685 gene, which encodes the homologue of the Shwachman-Bodian-Diamond syndrome (SBDS) protein, is located in the predicted exosome superoperon . The most common mutations associated with SDS include the polypeptide chain truncation K62X caused by the introduction of an in-frame stop codon (183–184TA → CT mutation) and a donor splicing site mutation, 258+2T → C, which causes premature truncation of the encoded protein by frameshift (84Cfs3). In addition, several point mutations across the entire sequence of the protein are also associated with SDS , with overall sequence similarities of 33%, 30% and 14% (BLOSUM62 substitution matrix), respectively, for domains I, II and III  is 3.3 Å. Again, superposition of individual domains results in lower main-chain r.m.s. differences: 1.3 Å (domain I), 1.0 Å (domain II) and 1.8 Å (domain III). These figures correspond to comparison of mthSBDS with [PDB: 1P9Q], but similar differences are found when comparison is made with the [PDB: 1T95] structure (main-chain r.m.s.d between the [PDB:1P9Q] and [PDB:1T95] structures is 0.4 Å). Thus, hereafter our discussion will be based on comparisons between mthSBDS and the [PDB:1P9Q] structure.Comparison of the two independent copies of mthSBDS present in the asymmetric unit with the available structures of afSBDS demonstrates differences in the relative orientation of the three domains Figure . The maiWe analyzed the changes in the relative orientations of domains I and III with respect to domain II between mthSBDS (molecule A) and afSBDS. When the central domains of the two molecules are superimposed, the observed rotations for domains I and III are ~16° and ~23°, respectively. The directions of the rotation axes are not coincident with those found between the two copies of mthSBDS in the asymmetric unit, indicating that apart from rotational rearrangement, the individual domains may undergo limited translational adjustment. Nevertheless, the hinge regions between the domains are the same. This observation is thus consistent with the domain rotations found for the two molecules of mthSBDS present in the asymmetric unit.M. thermautotrophicus were digested with nucleases and loaded onto mthSBDS-coupled NHS-activated affinity columns. The bound proteins were then eluted at acidic pH. The eluted proteins were separated in SDS-polyacrylamide gels, silver stained and the specific bands excised and examined by mass spectrometry . The largest contribution results from mode 11, which on its own contributes to 28% of the motions. Moreover, three among the first 10 non-trivial modes are sufficient to describe 64% of the conformational change between mthSBDS molecule A and afSBDS. In this case, a single mode (mode 8) contributes more than 40% to the inferred motion. Indeed, perturbation of the structure of mthSBDS molecule A along mode 8 causes the FYSH and C-terminal domains to rotate with respect to the central domain  is not found in solution. Under these circumstances, the formation of the dimer would be entropically disfavored.In the absence of a detailed understanding of the functions of SBDS proteins, we can only speculate about the potential roles of the structural rearrangements that we have demonstrated that these proteins might undergo. In this regard, we can gain some insight by considering the possible interaction partners of SBDS proteins. The three proteins interacting with mthSBDS protein that were identified in the present study by affinity chromatography are all from the large ribosomal subunit. These data give further support to an earlier study where the yeast SBDS protein (SDO1) co-purified with components of the 60S ribosomal particle . The datSince cell lysates were digested with RNAse prior to their loading onto the affinity columns, ribosomal proteins would be exposed to mthSBDS either as separate RNA-free proteins or as smaller protein-protein complexes. Therefore, the binding of these proteins to mthSBDS should be informative about the putative interacting surfaces between the mthSBDS and the ribosome. Within the large ribosomal subunit, the three interacting components of mth685 protein, namely the L1, L2 and L14, do not interact directly with each other . Structural observations reveal the intrinsic flexibility of SBDS proteins, a feature that we hypothesize to have direct implications for their functions. In keeping with previous results published for other SBDS homologs, we found, by means of affinity chromatography capture, that mthSBDS can interact with ribosomal proteins whereas RNA-SELEX experiments suggest it does not interact specifically with RNA. Involvement of the mthSBDS protein in the function and/or maturation of the translation machinery is possible, as previously suggested for SBDS proteins from other organisms  into three domains  and performed molecular replacement searches using these separate domains. The best solution was obtained for domain I of one of the two molecules (hereafter designated as molecule A) in the asymmetric unit. The solution for this domain was improved by rigid body refinement and was fixed during further molecular replacement searches. The second best solution was obtained for domain II of molecule B. At this stage rigid body refinement was applied to both domain solutions and was followed by 10 cycles of all-atoms restrained refinement using REFMAC .The atomic coordinates and structure factors have been deposited in the Protein Data Bank  (green) is superimposed with that of mthSBDS.Click here for file


Plasmodium falciparum–infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLβC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLβC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2βC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2βC2PF11_0521 best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLβC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLβC2 domain. DBL2βC2PF11_0521 binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2βC2PF11_0521 and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses. Plasmodium falciparum exports the protein PfEMP1 to the surface of parasitized erythrocytes for roles in immunoevasion and adhesion. The size and structural complexity of this diverse protein family have limited earlier studies of PfEMP1 biology to low throughput and semi-quantitative approaches. We developed a high throughput quantitative assay of PfEMP1 adhesion and used it to analyze structural features of domains that bind the putative cerebral receptor ICAM1, as well as to survey the acquisition of functional antibodies in malaria-exposed children and adults. In studies of the PfEMP1 repertoire of clone 3D7 parasites, a single specific domain bound ICAM1 strongly. PfEMP1 domains that bind ICAM1 strongly have conserved features, including conserved amino acids within otherwise highly variant flexible loops of the protein. While neutralizing antibodies against the PfEMP1–ICAM1 interaction were uncommon in Tanzanian children, such antibodies were common in east African adults, possibly explaining why immune adults rarely carry ICAM1-binding parasites. This high throughput platform will significantly accelerate studies of PfEMP1 binding domains and the corresponding antibody responses involved in protective immunity. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a virulence factor of the human malaria parasite P. falciparum. PfEMP1 variants are encoded by about 60 var genes per parasite, and have been implicated in the cytoadhesion of P. falciparum-infected erythrocytes (PE) to vascular endothelium The variant surface antigen var genes have been shown to bind specific ligands in vitro. For example, the CIDR1-α domain was implicated in binding CD36 Distinct domains of different Specific host receptors have been implicated in specific malaria syndromes  binds ICAM1 strongly. Our structural analyses suggest that DBL2βC2i.e., different bead regions), then bead regions of distinct intensity were incubated with lysates of COS cells expressing individual domains as GFP-fusion proteins, and washed extensively for fast immobilization/purification In our expression system, recombinant protein levels can be monitored during immobilization/purification on immobilized anti-GFP antibodies by fluorescence, as we previously described using the multi-well plate format PF11_0521 domain among the 16 domains tested bound to ICAM1 at high levels . In loop 3, a 3-amino acid motif containing a hydrophilic residue-hydrophobic residue-hydrophilic residue, is completely conserved in binding domains. The hydrophobic residue in this motif is Ile with a single conservative exception , and appears to be in close contact with the ICAM1 molecule in the model PF11_0521 domain. Similarly, in FCR3 strain parasites, 3 non-binding domains carry Ala or Leu at this position. However, the non-binding domains with the Ala or Leu residue in loop 4 contain multiple substitutions in other conserved regions and positions, which may explain their non-binding status.In previous analyses, an Ala or Leu residue was observed in position 3 of loop 4, in all but one ICAM1 binding domain (the exception being the FCR3/IT var1 DBL2βC2 domain where His is present) . In the 286 with Tyr in loop 4. The substitution does not introduce any amino acid residue clashes, and may provide an additional intra-domain hydrogen bond to R113 (data not shown). We infer that the loss of binding to ICAM1 is not due solely to substitutions of Ala or Leu in loop 4, but results from combinations of substitutions that involve this and other residues in the protein. We are currently testing this hypothesis using our quantitative BioPlex approach and site-specific mutagenesis. Other residues that we predict may have an effect on the domain structure or ligand binding are indicated in red in Generally, ligand interactions involving substantial surfaces of amino acid residues are not significantly altered by substituting a single residue that participates in binding, so long as the substitution fits into the structure without clashes and does not affect structural integrity  Our sequence and binding analyses indicate that structural DBLβC2 domain is larger than previously suggested PF11_0521 domain  in assays that measured reversal of adhesion  adhesion with immune sera We tested inhibition of ICAM1 binding to DBL2βC2S adults . Pooled adhesion , indicatPF11_0521 domain . Neutralizing activity did not increase significantly in the first 2 years of life, although a trend to increasing activity was observed after 100 weeks of life. The same trend appeared in our longitudinal cohort study of 7 patients described above  in a system that is well-suited for folding of transmembrane disulfide-rich proteins. Therefore, all recombinant domains have a high probability of folding similarly well. Because one of DBLβC2 domain variants clearly demonstrated binding activity, we assume that other variants that did not bind were properly folded but do not function as ICAM1 ligands. Nevertheless, since every protein is unique, false negatives can not be excluded completely without direct proof of correct folding by methods like X-ray or NMR, which are outside the scope of this study. Our findings also suggest that the structural/functional DBLβC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLβC2 domain PF11_0521 appear sporadically in the first 2 years of life  and CD36 (data not shown) in the pAdEx expression system also supported binding in the pHisAdEx expression system. Primers used for cDNA amplification using 3D7 genomic DNA are shown in supplementary All constructs were cloned into the pAdEx vector described earlier 25 µg of anti-GFP antibody  was coupled to 200 µl of each different BioPlex bead region (Bio-Rad)  as described by the manufacturer, then resuspended in PBS containing 1 mg/ml BSA, 0.05% Tween-20, and 0.02% sodium azide (PBS-TBN buffer). Anti-GFP-coupled beads were incubated for 2 hours at 4°C with COS-cell lysates containing expressed domains, washed in PBS-TBN, and used in ligand binding experiments. These beads are designated as DBLβC2-coupled beads.All DBLβC2-coupled beads were mixed together in quantities of ∼40–60 beads of each bead region (beads with distinct fluorescence intensity) per µl. 50 µl of bead mixtures were transferred into individual wells of HTS 96-well plates (Whatmann) that were pre-incubated with PBS-TBN for 30 minutes. Beads were washed in wells 3 times with PBS-TBN and incubated with different concentrations (20 – 0.1 µM) of ICAM1-human Fc receptor . After 1 hour incubation at room temperature (RT) at constant rotation at 600 rpm, beads were washed in PBS-TBN and incubated in similar fashion with 1∶10 diluted biotinylated anti-ICAM1 monoclonal antibody (mAb) RR-1  followed by 1 hour incubation with 1∶250 diluted streptavidin-phycoerythrin (SA-PE) fluorescent molecules . Also, in some experiments  we used anti-human IgG coupled to phycoerythrin  to detect bound ICAM1-human Fc and obtained almost identical results. After a final wash, 96-well plates were transferred into the BioPlex apparatus (Bio-Rad) to quantify ICAM1 binding (measured in phycoerythrin channel) to the individual DBLβC2 domains. For negative controls, lysates prepared from mock-transfected cells, and from pAdEx vector transfected cells, were also coupled to beads. These control beads were mixed with the DBLβC2-coupled beads and assayed simultaneously. The pAdEx vector produces GFP-fusion proteins that contain an irrelevant peptide of 37 amino acids in the extracellular domain.PF11_0521, ICAM1-Fc (1 µg/ml) was incubated with various concentrations of mAb My13 (Axxora) for 1 hour at room temperature and then used to bind to the mixture of His-DBL2C2PF11_0521 and HisAdEx (negative control) coupled beads as described above using RR1 mAb for detection.To confirm the specificity of ICAM1 binding to DBL2C2For binding inhibition assays DBLβC2-coupled beads were pre-incubated for 1 hour at RT with various plasma samples diluted (1∶5) in PBS-TBN. The beads were then assayed in the same fashion as for the binding assay described above using RR1 mAb for detection. For binding-reversal assays, the binding assay was performed as described above, except that the beads were incubated with various plasma samples diluted (1∶5) in PBS-TBN for 1 hour at RT after the reaction with SA-PE, and then washed just prior to quantification in the BioPlex apparatus.Human plasma samples used in these studies were collected from East African donors under protocols approved by relevant ethical review committees. Study participants provided written informed consent before donating samples, and included adult males from Kenya PF11_0521 domain-coupled beads were washed in wells 3 times with PBS-TBN and incubated with children plasma samples at 1∶100 dilution in PBS-TBN. After 1 hour incubation at room temperature at constant rotation at 600 rpm, beads were washed in PBS-TBN and incubated in similar fashion with 1∶250 diluted anti-human IgG coupled to phycoerythrin (Jackson ImmunoResearch) for 1 hour. Signal was measured in the BioPlex apparatus to quantify bound IgG. Signals obtained for beads coated with protein expressed by pAdEx vector were used as negative controls and were subtracted from signal obtained with DBL2βC2PF11_0521 domain-coupled beads. Also, pooled samples of non-immune US plasma (n = 5) and immune plasma from adults living in malaria endemic region (n = 5) were used as additional negative and positive controls, respectively.DBL2βC2Figure S1Scheme of high throughput functional assays based on BioPlex technology. (A) Scheme of immobilization-purification of PfEMP1 domains on BioPlex beads. (B) Scheme of detection of receptor-binding domains and inhibition of receptor binding. Up to 100 different bead regions (colors) each coupled to 1 protein can be mixed together in 1 well for interaction with ligand. Various blocking reagents (like antibodies) can be added or pre-incubated before addition of a ligand. All beads from each well then are taken up into BioPlex machine, each bead region is classified by color (1st laser). Simultaneously, signal from detection molecule is measured by 2nd laser for each bead region (protein).(0.07 MB DOC)Click here for additional data file.Figure S2Alignment of 3D7 DBLβC2 domains and FCR3/IT DBLβC2 domains. FCR3/IT DBLβC2 domains are designated as var1 through var44. Numbering of residues begins with the first N-terminal residue shown in the figure for FCR3/IT domains, or with the first residue of the recombinant 3D7 domains used for assays in this study. All 3D7 domain sequences contain the minimal binding region identified previously in a semi-quantitative assay (0.19 MB DOC)Click here for additional data file.Figure S3Model of Ile and Val amino acid residues in “loop 3” of DBLβC2 domain interacting with ICAM1. Illustration of interaction and amino acid residue substitution were created using DBLβC2::ICAM1 complex modeled in (0.31 MB DOC)Click here for additional data file.Figure S4Alignment of two DBLβC2 domain sequences (bottom) and three DBLβ domain sequences (previously described as lacking adjacent C2 domains) with surrounding sequences (top). Color font: Orange - cysteines not present in any sequence in (0.06 MB DOC)Click here for additional data file.Table S1Primers for PCR amplification of 3D7 DBLβC2 domains.(0.05 MB DOC)Click here for additional data file.Table S2PF11_0521 domain.Forward primers for PCR amplification of five 3D7 DBLβC2 domains in the same boundaries as active DBL2βC2(0.03 MB DOC)Click here for additional data file.


Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini.The Hedgehog (Hh) signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA), and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria.We performed extensive genome searches such as the cnidarian hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are found in many protists indicates that this gene family must have arisen in very early eukaryotic evolution, and gave rise eventually to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins are three families of Hint domain proteins that evolved in parallel in eukaryotes.In Cnidaria and nematodes the evolution of  Drosophila melanogaster has a single hedgehog (hh) gene, while mammalian genomes contain three paralogous genes, Sonic Hh (Shh), Desert Hh (Dhh), and Indian Hh (Ihh) [hh genes are present due to an extra round of genome duplication during evolution of ray-finned fish [Caenorhabditis elegans no bona-fide hh is present, i.e. there is no gene that encodes both the N-terminal signalling domain as well as the C-terminal Hint domain. Instead ten genes encoding the C-terminal autoprocessing domain are found that, however, have N-terminal regions very distinct from Hh. Furthermore, a large number of additional genes are found that encode only these new N-terminal domains and lack the C-terminal autoprocessing domain. Overall, these genes can be grouped into four families that have been named quahog (qua), warthog (wrt), groundhog (grd) and ground-like (grl) and are collectively referred to as hh-related genes [C. elegans Hh-related proteins can add a cholesterol moiety to the N-terminus analogous to Hh, since there are sequence differences in the SRR equivalent region. Therefore, this region of the Hh-related proteins was named ARR (adduct recognition region) [The Hedgehog (Hh) signaling pathway has been shown to be of fundamental importance for patterning and cell proliferation in animal development (for review see -4). MutaHh (Ihh) . In zebrned fish ,11. The ned fish . C-termined fish -16. In ted genes -19. At p region) ; here we region) .C. elegans hh-related genes were not found in vertebrates and flies using blast searches, giving rise to the notion that these genes were perhaps derived from hh in early nematode evolution [Monosiga ovata, but its N-terminal region is distinct from Hh and other Hh-related proteins, instead sharing sequence similarity with cellulose-binding domains (CBD) [Nematostella vectensis [C. elegans hh-related genes, we had already performed cursory searches of the genome of the parasitic nematode Brugia malayi and found that it also contains several hh-related genes [N. vectensis [B. malayi and Trichinella spiralis as well as the NCBI protein, DNA and EST databases to find additional hh and hh-related genes that may shed light on the evolution of these genes. In these searches we found a previously described gene from the fungus Glomus mosseae that shares sequence similarity with Hh through the Hog domain [The N-terminal domains of the volution ,18. Recens (CBD) . Choanofns (CBD) ,24 and tectensis ,26, indiectensis , indicated genes . Here weectensis , the nemg domain , but hasg domain . Furtherg domain -32. Receg domain ,33. Seveg domain -37. Our qua, one hog-only, ten wrt, 17 grd, and 32 grl ORFs from C. elegans, three of which are pseudogenes [hh-related genes in the related nematode Caenorhabditis briggsae [hh-related genes here, because C. briggsae wrt-8 is the same locus as wrt-4. To retrieve sequences from other species we used selected Hh, WRT, QUA, GRD and GRL protein sequences as queries for tblastn and blastp searches at Stellabase, the DOE Joint Genome Institute, The Genome Sequencing Center at the Washington University School of Medicine, The Institute for Genomic Research (TIGR), and NCBI (see Methods). The recovered sequences were aligned to sequences that we had assembled previously [B. malayi wrt-4). In the case of ESTs it was often possible to assemble several ESTs into contigs, but in most instances ORFs derived from ESTs lack either N-terminus and/or C-terminus. Considering also that the various genome projects are are in different states of completion, the nomenclature given here to the ORFs should be considered preliminary. After correction of the ORFs multiple sequence alignments of the different protein domains were made and used for phylogenetic analyses using Neighbor Joining and Maximum Likelihood. We also prepared protein sequence logos of the Hog domains of Hh and nematode Hh-related genes to aid with the analysis of more divergent Hog domains , two without a Hog domain  and one whose C-terminus is presently unknown (Bm wrt-4)  Table , Bm wrt-rt-6 EST  lacks thromosome , howeverB. malayi, Bm grd-5, that is co-orthologous to Ce grd-5 and grd-10   are clearly hh genes . While its Qua domain is rather divergent, the cysteine residues are all conserved . They share sequence similarity upstream of the Hog domain with six conserved cysteine residues. In addition, XC Xhog5 also has sequence similarity to the upstream regions of Ts Xhog1 and Xhog 2 , but no Hog domain follows  and the apicomplexans Cryptosporidium muris and Cryptosporidium parvum (blast prob. of aCp Hog: 4e-08). Their Hog domains contain motifs J and K, although in a few cases the cysteine has been replaced with a serine in motif J  and Physcomitrella patens one sequence each with a Hog domain; in the red algae Chondrus crispus two sequences (blast prob. of rCc Hog: 1e-10); in Griffithsia japonica one sequence (blast prob.: 3e-08); in Porphyra haitanensis (blast prob. of rPh Hog: 4e-07) and Porphyra yezoensis two Hog domain ORFs each; and in Gracilaria changii six ORF fragments (blast prob. of rGc Hog1: 3e-12). Those moss and red algae Hog domains that are not truncated have motifs J and K, although the cysteine residue in motif J has been changed to serine, threonine, or aspartate. Alignment of rPy and rPh Hog2 revealed conserved sequences upstream of the Hog domain, however, these two sequences are relatively closely related so this conservation is not surprising , however, it has a much better match to Hh Hog domains (blast prob.: 3e-24) than other non-metazoan Hog domains, and we could not find any match to rice genomic sequences. Therefore, this sequence may come from a contaminating organism and is designated as species XX here.Further Hog sequences were found in red algae and mosses Figure : In the g Figure . Blast sg Figure . The P. Bigelowiella natans (blast prob.: 9e-10), from the cryptophyte Guillardia theta (blast prob. of crGt Hog1: 6e-11 to Hog of Mo hoglet), and from the jakobid Jakoba libera (blast prob. jJl Hog1: 2e-05) , and costa are missing [C. elegans [N. vectensis [C. elegans in the relatively well sequence genomes of N. vectensis and T. spiralis. Using reciprocal blast searches, we have attempted to identify these components of the pathway in Nematostella and Enoplea . No match could be found for the cDNA sequence from rice (pOs AK110392) in the genomic sequence, but ESTs recovered from other plants support the notion that VWA-Hint proteins exist in plants.During the tblastn searches ESTs and ORFs from non-Hog genes such as inteins were discovered, usually in the non-significant zone at the bottom of the results lists. One group of genes attracted our attention, because upon closer inspection it became apparent that these genes had an amino-terminal domain comprised of a VWA domain followed by a region that has good similarity to the first part of the Hint domain, i.e. in particular motifs A and B Figure . This obSaccharomyces cerevisiae), and in Metazoa. Multiple independent losses in different lineages seem the most likely explanation for this absence.The VWA-Hint proteins do not seem to have a signal peptide for secretion. The VWA domain is located at the N-terminus of the proteins, although in four cases a Ubox precedes the VWA domain Figure , 18, 19.Our searches also revealed a group of proteins from bacteria that had a Hint-like domain at their C-terminus and shared some weak sequence similarity in their N-terminal region . Therefore, some additional hh-related genes might still be forthcoming. But the present survey shows that members of the qua, wrt, grd and grl gene families are all present in B. malayi. Only a representative of a grd gene with a Ground domain has so far not been found. The phylogenetic analyses show that while there are some instances of direct orthology between B. malayi and Caenorhabditis genes, in many instances, in particular for the grl genes, the relationship is not clear and in fact suggests that independent diversification occurred in these two Chromadorea branches. This shows that these gene families have been actively evolving in nematodes.Hh genes are present in deuterostomes as well as in several different protostome phyla such as molluscs, annelids, and arthropods Figure . HoweverXiphinema sp. and T. spiralis a strikingly different pictures emerges. In both species we find both a hh gene as well as several hh-related genes. In T. spiralis we also find a quahog gene, and – based on the phylogenetic analyses – some of the Xiphinema genes could also be quahog genes. Two T. spiralis and one Xiphinema protein share a new motif (Enop motif) upstream of the Hog domain that appears to be specific to Enoplea nematodes. However, there are also a number of instances of N-terminal sequences that are very short. Several of these proteins cluster with the "Enop" proteins in the phylogenetic analyses, suggesting that they diverged from a common ancestor. However, two proteins with short N-terminal regions (Ts Xhog3 and XC Shog2) are rather divergent and do not reliably fall within the clade of nematode-specific Hog proteins ("Nema-Hog" proteins) in phylogenetic analyses. In particular Ts Xhog3 lacks the conserved cysteine pair usually found in Nema-Hog domains, and it shares motifs K and L with Hh Hog domains, indicating it could be derived from a Hh protein. Therefore, while these genes could have diverged from hh or Nema-Hog genes, it may also be possible that the represent ancestral genes that were lost in Chromadorea. In conclusion, we think that there were probably at least three different types of Hog genes in the common ancestor of Enoplea and Chromadorea, one hh gene, one quahog gene and one gene which give rise to the wrt/grd branch in Chromadorea and the Ts Xhog1/2 branch in Enoplea. But possibly up to five Hog genes could have existed in the common ancestor. The proliferation into further distinct groups such as wrt, ground and ground-like appears to have happened later in a branch specific manner.In the more distantly related Enoplea nematodes wrt and ground families. The presence of the rather short N-termini in Enoplea suggests that these regions have evolved and diverged through mutations, rather than by acquisition of a new domain. The threonine-rich stretch in XC Thog is very likely the result of polymerase slippage, though it is striking that this feature has evolved separately also in the choanoflagellate Hoglet protein [grl-23 has a 176 residue long stretch upstream of the Ground-like domain containing 125 glycine residues. In conclusion, most of the observed variability in the N-terminal domains of nematode Hh-related proteins is probably the result of sequence divergence from a progenitor, rather than acquisition of new domains. Loss of N-terminal domains in the case of C. elegans Hog-1, as well as loss of Hog domains did occur however.Many different N-termini now exist in Nema-Hog proteins. Two possible mechanisms can explain this diversity: Either acquisition of new N-terminal domains, or divergent evolution of existing N-terminal domains. A relatively good case can be made that all Wart, Ground and Ground-like domains arose from a single common ancestor based on weak sequence similarities between the motifs . This re protein . It is aT. spiralis has a hh gene, but apparently lacks several components of the Hh pathway, such as Smoothened. Particularly noteworthy is that the components that appear to be missing are the same as in C. elegans. This would suggest that the signaling pathway was modified by loss already before the split of Enoplea and Chromadorea, even though hh was maintained in Enoplea. While one could imagine that Hh could be maintained in an animal parasite such as T. spiralis to affect host cells, this is very unlikely in the case of the plant nematode Xiphinema. It implies that Hh has an important function even in the absence of Smoothened, and it refutes the hypothesis that the Nema-Hog genes evolved directly from hh concomitantly with the other changes in the Hh pathway.A surprising observation is the fact that N. vectensis and A. millepora we find bona-fide hh genes that have a Hedge and a Hog domain. Another gene is well conserved between N. vectensis and A. millepora and has a distinct, novel secreted N-terminal domain. Two further Hh-related proteins in N. vectensis have yet other, distinct N-termini. The upstream region of the two closely related genes retrieved from Hydra do not share any similarity with those in Nematostella, indicating divergent evolution. No sequence similarity of these new N-terminal motifs has been found outside Cnidaria.In Cnidaria we also encounter a complex situation with both Hh and Hh-related proteins. Both in hh-related genes from Cnidaria are however distinct from those in nematodes, since the phylogenetic analyses of the Hog domains does not show them to be closely related. Therefore, we would like to suggest that – as in the case of the nematode hh-related genes – the Cnidarian N-terminal domains have evolved from common ancestors by divergent evolution rather than by domain acquisition.The The case of Nv 200640 is perhaps a special exception. In this protein we find an N-terminal Hedge domain fused to a large extracellular protein that contains a VWA domain as well as CA and EGF repeats, but it clearly lacks a Hog domain. The VWA domain is a 200 residue long domain first identified in von Willebrand Factor ,45. VWA hh and hh-related genes. Did the hh-related genes evolve twice independently from a hh precursor in each lineage? This is certainly the most parsimonious hypothesis. Nonetheless, in an alternative scenario, a hh and a hh-related gene could have been present in the common ancestor of eumetazoa, and the hh-related gene would have given rise to the cnidarian and nematode hh-related genes. For this hypothesis to be true, we would have to postulate three separate losses of hh-related genes: in deuterostomes, in lophotrochozoa, and in arthropods. While this seems rather unlikely, we do observe many losses of Hog genes in various branches of eukaryotes, as well as loss of the Hog domain only in a number of nematode genes so that such a series of losses may not be totally impossible.We have recovered a substantial number of Hog domain proteins from many diverse groups of eukaryotes, mostly protists, such as red algae, moss, alveolates , cryptophytes, jakobids, haptophytes, cercozoa and Glomeromycota fungi. While some of these Hog sequences are quite divergent, they are invariably most closely related to Hog domain proteins from animals, and not to inteins, such as those found in fungi, or to BIL or Vint domains. Given the widespread occurrence in many of the major groups of eukaryotes  with its distinct features was already present in the progenitor of two very different nematode branches it may be possible that both Hh and some other Hog domain protein was already present in protostomes before the emergence of nematodes and was lost in other lineages such as arthropods. The finding of multiple Hog domain proteins in Cnidaria raises the possibility that multiple distinct types of Hog domain proteins also existed in ancestral Eumetazoa. Snell et al. (2006) suggested that a precursor of a Hedge domain fused to a Hog domain in early Metazoan evolution. However, our discovery that an Ur-Hog gene probably existed in the progenitor of eukaryotes makes if feasible that Hh evolved from an ancestral Hog gene without domain shuffling. In eukaryotes, we now know that at least three different types of Hint domains evolved in parallel: Hog, Vint, and inteins. At present we do not know the origin of the Hog and Vint domains, but perhaps new Hint domains from bacteria, such as described here and by Dassa and Pietrokovski [We find that the evolution of Hh is more complex than anticipated, and that this gene family is not simply derived from an intein in early metazoan evolution. Both in Cnidaria and nematodes parallel evolution between rokovski  will sheB. malayi sequences were searched at TIGR [B. malayi is deposited regularly into the GSS division of GenBank. This sequencing effort is part of the International Brugia Genome Sequencing Project and is supported by an award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. ESTs, in particular nematode ESTs, were searched at NCBI [T. spiralis genome was searched using the GSC blast server at The Genome Sequencing Center of the Washington University School of Medicine [N. vectensis sequences were searched at Stellabase [Naegleria gruberi, Physcomitrella patens and Monosiga brevicollis were searched at the JGI [Procedures for retrieving and analyzing sequences have been detailed in Hao et al. 2006 and Mukherjee and Bürglin 2007 ,48. Brie at TIGR . Prelimi at NCBI . The nem at NCBI . Many of at NCBI . The T. Medicine . N. vectellabase ,28, and ellabase . Additio the JGI . Zebrafi the JGI ,57. The  the JGI ,58. Manu the JGI  and PPCM the JGI . ESTs re the JGI .Sequences were added to an existing database of Hh and Hh-related proteins , and areAll research was carried out by TRB and the manuscript was written by TRB.Multiple sequence alignment of Hog domains used for the protein sequence logos. Multiple sequence alignment in this and subsequent figures was carried out using first MUSCLE and imported subsequently into Clustal_X. Color coding was modified from default Clustal_X color coding by marking all cysteine residues in yellow, small hydrophobic residues in light blue and large hydrophobic residues in cyan blue. The conserved motifs, as well as the C-terminal SRR or ARR region are indicated in the alignment. The two conserved cysteine residues found in the Hog domain of nematode Hh-related proteins are indicated with red arrows.Click here for fileFull image of the protein sequence logo of aligned Hog domains shown in Figure Click here for fileProtein sequence logo of nematode Hog domains. Protein sequence logo generated from nematode Hog domains shown in the multiple sequence aligment of Additional file 1. This logo is in register with the Hh Hog domain logo of Additional file 2. The two conserved cysteine residues specific to nematode Hh-related proteins are indicated with red arrows.Click here for fileMultiple sequence alignment of Wart domains. Wart domains were aligned and visualized in Clustal_X as described in Figure Click here for filePhylogenetic tree analysis of Hog domains using Neighbor joining. Neighbor joining tree without protist sequences. The Hog domain of the fungal gene GmGIN1 was used as outgroup.Click here for filePhylogenetic tree analysis of Hog domains using Maximum likelihood. Maximum likelihood tree of the same sequences as in Additional file 5 with GmGIN1 as outgroup.Click here for filewrt genes (arrow). GmGIN1 was used as outgroup. Note: Apart from Figure C. tropicalis was used as outgroup [Neighbor joining tree of Hog sequences which were truncated at the N-terminus. Hog sequences were truncated at the N-terminus to have the same size as the Pt wrt sequence fragment. This analysis shows that Pt wrt clusters with the outgroup  and gaveClick here for fileMultiple sequence alignment of Ground and Ground-like domains. Alignment of nematode Ground and Ground-like domains.Click here for fileSequences used in the analysis. List of sequences, accession numbers, notes, predicted signal peptide cleavage sites and protein sequences used in this analysis.Click here for fileMultiple sequence alignment of "Hedge" domain containing proteins and Hedgehog proteins. Note that Os hhlike and NV 200640 do not line up in the Hog domain region.Click here for fileMultiple sequence alignment of Quahog proteins. Alignment of nematode Quahog proteins.Click here for fileStructure of the Nematostella vectensis genomic assembly around Nv 239508. Current assembly of the genomic region around Nv 239508. Color arrows indicate duplicated regions. N gap indicateds two regions with unknown sequence. The green area shows the ESTs found mapping to this region. The CAGN20453 correpsonds to Nv 239508. The yellow area shows regions of sequence similarity, i.e. hydrolase domain, Hog domain, and Reverse transcriptase. CAGN20453 is not sequenced fully, but the 3' read has been mapped to the right side, since the 3' untranslated region matches better to the 2. repeat of the duplication due to some indel differences. However, as will be noted, the final resulting transcript (shown at bottom) would be rather unusual, as it would splice over another gene, i.e. the hydrolase, which is also supported by an EST. Hence, the genomic organization and gene structure in this region could be subject to change, especially given the unsequenced areas.Click here for filePredicted protein structure of Nv 200640. Protein motif prediction of the SMART server was used to analyse the ORF Nv 200640, and the different types of conserved motifs found are indicated.Click here for fileMultiple sequence alignment of non-metazoan Hog proteins. Alignment of non-metazoan Hog proteins including also the ones which are only based on EST fragments.Click here for fileMultiple sequence alignment of the Hint region of VWA-Vint proteins with Hog domains of Hh proteins.Click here for fileMultiple sequence alignment of bacterial proteins with a novel type of Hint-like region. Full length multiple sequence alignment of bacterial proteins. Motifs A and B are marked, as well as a small region with some similarity to the beginning of motif F in BIL-As. Note: the conserved upstream regions may be secreted, since some of the sequences have bacterial signal peptides for secretion. Species and the accession number for the sequences are: bSa_STIAU_1829: Stigmatella aurantiaca DW4/3-1 (ZP_01466308); bMx_MXAN_6253: Myxococcus xanthus DK 1622 (YP_634382); bRM_MED297_11140: Reinekea sp. MED297 (ZP_01113290); bPl_plu1731: Photorhabdus luminescens subsp. laumondii TTO1 (NP_929012); bSp_Draf4685: Serratia proteamaculans 568 (ZP_01534811); bYi_YintA_01002283: Yersinia intermedia ATCC 29909 (ZP_00833384).Click here for fileMultiple sequence alignment of the Hint region of the bacterial proteins with a novel Hint-like domain with Hog domains. Multiple sequence alignment of the bacterial Hint domains from Additional file 16 with Hog domains from animals. Note the roughly similar length.Click here for file


We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis–trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits. Escherichia coli-expressed monomeric form of the protein (ecCTLA-4), which unexpectedly formed a misfolded dimer and both amyloid and amorphous aggregates under largely physiological conditions. Our work provides new insights into the stability of immunoglobulin folds and the process by which they form disease-inducing amyloid-like deposits.Immunoglobulin (Ig) light-chain protein deposition diseases arise when Ig domains form pathology-inducing aggregates; these can occur in a variety of organs but most frequently in the kidney.Fo − Fc map shows clearly the strand-swapped density. The GG′ strands of one domain replace the GG′ strands of a neighbour, and vice versa, forming a compact dimeric unit. A structural comparison with the native, non-strand-swapped crystal structures of B7-complexed CTLA-4et al., manuscript in preparation) which forms a quite different disulfide-linked dimer, revealed that cis–trans isomerisation of the prolines at positions 101 and 103 relieves peptide bond strain in ecCTLA-4 seemingly driving the strand swap of the C-terminal β-strands  V-set domain, was engineered by mutating the cysteine at position 122, which forms a disulfide between the C-terminal membrane-proximal linker regions of the protein, to a serine. As expected, this construct was expressed as a monomer see , and its-strands b and c. The strand swap seen in our CTLA-4 structure is strikingly similar to that found for CD47et al. in which 2,2,2-trifluoroethanol (TFE) is used to create mildly denaturing conditions and so stimulate aggregation.To assess the amyloid-forming propensity of the monomeric and strand-swapped dimeric forms of CTLA-4, we employed an assay established by Wright et al.What is the molecular nature of these aggregates? The ultrastructure was visualised by negative stain electron microscopy and is displayed in et al.,When calculating the strand-swapping capacity of the IgSF domain as defined by Bennett et al. that immunoglobulin domains of similar sequence aggregate much faster and to a greater extent into amyloid-like structures than domains of dissimilar sequence: swapping β-strands of dissimilar sequences would effect imperfect structural complementation and decreased stability.in vivo, being most abundant in bone marrow, blood and lymph nodes.Domain swapping of the type we have observed provides an explanation for the observation made by Wright It is noteworthy that the two-β-sheet DEBA–GFCC′C″ topology characteristic of IgSF domains appears independently in cytokine receptors, fibronectin, cadherins, transcription factors and bacterial cytosolic domains, indicating that it is among the most successful folding topologies. In spite of this, there are now four examples of IgSF domains exhibiting intrinsic metastability: CTLA-4, CD47, the llama antibody heavy chain and CD2.2x44.Coordinates and structure factors for the ecCTLA-4 strand-swapped dimer have been deposited with Protein Data Bank accession code